Prodrugs of excitatory amino acids

ABSTRACT

This invention relates to synthetic excitatory amino acid prodrugs and processes for their preparation. The invention further relates to methods of using, and pharmaceutical compositions comprising, the compounds for the treatment of neurological disorders and psychiatric disorders.

This invention provides synthetic excitatory amino acid prodrugs(compounds of Formula I) and processes for their preparation. Theinvention further relates to methods of using, and pharmaceuticalcompositions comprising, the compounds of Formula I for the treatment ofneurological disorders and psychiatric disorders.

BACKGROUND OF THE INVENTION

Treatment of neurological or psychiatric disorders, such as anxietydisorders, have been linked to selective activation of metabotropicexcitatory amino acid receptors. For example,(+)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid isdisclosed as an active mGluR2 receptor agonist in U.S. Pat. No.5,688,826 (the '826 patent), issued Nov. 18, 1997. Additionally,(+)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid isdisclosed as an active mGluR2 receptor agonist in U.S. Pat. No.5,958,960 (the '960 patent), issued Sep. 28, 1999.

The present invention provides for prodrug forms of mGluR2 receptoragonist compounds, which enhance the in vivo potency of the respectiveparent compound and produce higher oral exposure of the parent compound.Compounds of the present invention represent the best approach formaintaining the safety and efficacy of previously disclosed mGluR2receptor agonists with increased oral bioavailability.

Synthetic excitatory amino acid prodrugs and processes for theirpreparation are disclosed in PCT Application Serial Nos. PCT/US01/45866and PCT/US02/00488.

SUMMARY OF THE INVENTION

The present invention provides a compound of Formula I

wherein

-   -   A is H-(Q)_(p)-;    -   Q is independently selected, each time taken, from the group        amino acyl;    -   p is an integer from 1 to 10;    -   X is O, S, SO, SO₂, or CR³R⁴;    -   R³ is fluoro, X′OR⁵, SO₃H, tetrazol-5-yl, CN, PO₃R⁶ ₂, hydroxy,        or NO₂, and R⁴ is hydrogen; or R³ and R⁴ each represent fluoro;        or R³ and R⁴ together represent ═O, ═NOR⁷, or ═CR⁸R⁹; or one of        R³ or R⁴ represents amino and the other represents carboxyl; or        R³ represents N₃, (CH₂)_(m)COOR^(5a), (CH₂)_(m)PO₃R^(6a) ₂,        NHCONHR^(5b), or NHSO₂R^(5c), and R⁴ represents hydrogen; or R³        and R⁴ together represent ═CHCOOR^(5b), ═CHPO₃R^(6a) ₂, or        ═CHCN;    -   X′ represents a bond, CH₂, or CO;    -   m is an integer from 1 to 3;    -   R⁵, R^(5a), R^(5b), R^(5c), R⁷, R⁸, and R⁹ are independently a        hydrogen atom; an optionally substituted (1-6C) alkyl group; an        optionally substituted (2-6C) alkenyl group; an optionally        substituted (2-6C) alkynyl group; an optionally substituted        aromatic group; an optionally substituted heteroaromatic group;        a non-aromatic carbocyclic group; a non-aromatic heterocyclic        group; a non-aromatic monocyclic carbocyclic group fused with        one or two monocyclic aromatic or heteroaromatic groups; or a        non-aromatic monocyclic heterocyclic group fused with one or two        monocyclic aromatic or heteroaromatic groups;    -   R⁶ and R^(6a) independently represent hydrogen or a (1-6C)alkyl        group;    -   R¹⁰ is hydrogen or fluoro; and

R¹¹ is hydrogen, fluoro, or hydroxy;

or a pharmaceutically acceptable salt thereof.

The present invention also provides a compound of Formula I whereinsubstiutents are defined as above, provided that the compound is not onein which X is CR³R⁴ wherein R³ is fluoro and R⁴ is hydrogen, p is 1, andQ is L-alanyl; or a pharmaceutically acceptable salt thereof.

The present invention also provide a compound of Formula I wherein A isH-(Q)_(p)-; Q is independently selected, each time taken, from the groupamino acyl; p is an integer from 1 to 3; X is O, S, SO, SO₂, or CR³R⁴;R³ is fluoro or hydroxy, and R⁴ is hydrogen; or R³ and R⁴ togetherrepresent ═O; R¹⁰ is hydrogen or fluoro; and R¹¹ is hydrogen, fluoro, orhydroxy; or a pharmaceutically acceptable salt thereof.

The present invention also provides a compound of Formula I wherein A isH-(Q)_(p)-; Q is L-alanyl; p is 1; X is CR³R⁴; R³ is fluoro and R⁴ ishydrogen; R¹⁰ is hydrogen; and R¹¹ is hydrogen; or a pharmaceuticallyacceptable salt thereof.

It will be appreciated that the compounds of Formula I contain at leastfour asymmetric carbon atoms. The present invention includes allstereoisomeric forms of the compounds of Formula I, including each ofthe individual enantiomers and mixtures thereof such as prodrug forms ofcompounds disclosed in the '826 patent such as, for example,1SR,4RS,5RS,6RS-4-amino-(2-sulfonylbicyclo[3.1.0]hexane)-4,6-dicarboxylicacid.

A further aspect of the present invention provides for a pharmaceuticalformulation comprising in association with a pharmaceutically acceptablecarrier, dilutent, or excipient, a compound of Formula I, or apharmaceutically acceptable salt thereof.

A further aspect of the present invention provides for a method foraffecting the cAMP-linked metabotropic glutamate receptors in a patient,which comprises administering to a patient requiring modulatedexcitatory amino acid neurotransmission a pharmaceutically-effectiveamount of a compound of Formula I. This invention also provides for ause of a compound of Formula I for the manufacture of a medicament foraffecting the cAMP-linked metabotropic glutamate receptors in a patient.

A further aspect of the present invention provides for a method ofadministering an effective amount of a compound of Formula II thatcomprises administering to a patient requiring modulated excitatoryamino acid neurotransmission a pharmaceutically effective amount of acompound of Formula I. This invention also provides for a use of acompound of Formula I for the manufacture of a medicament foradministering an effective amount of a compound of Formula II.

A further aspect of the present invention provides for a method fortreating a neurological disorder in a patient that comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of Formula I. Thisinvention also provides for a use of a compound of Formula I for themanufacture of a medicament for treating a neurological disorder in apatient.

A further aspect of the present invention provides for a method fortreating a psychiatric disorder in a patient that comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of Formula I. Thisinvention also provides for a use of a compound of Formula I for themanufacture of a medicament for treating a psychiatric disorder in apatient.

Compounds of Formula I may be made by a process that is analogous to oneknown in the chemical art for the production of structurally analogousheterocyclic compounds or by a novel process described herein. Suchprocesses and intermediates useful for the manufacture of a compound ofFormula I as defined above are illustrated by the following proceduresin which, unless otherwise specified, the meanings of the genericradicals are as defined herein.

The present invention provides a process for preparing compounds ofFormula I comprising acylating a compound of formula (ii):

with a corresponding amino acyl of Formula IIIPg^(N)-A-  (III)

-   -   wherein Pg^(N) is a nitrogen-protecting group and A is as        defined above;    -   whereafter, for any of the above procedures, when a functional        group is protected using a protecting group, removing the        protecting group;    -   whereafter, for any of the above procedures: when a        pharmaceutically acceptable salt of a compound of Formula I is        required, reacting the basic form of such a compound of Formula        I with an acid affording a pharmaceutically acceptable        counterion; or for a compound of Formula I which bears an acidic        moiety, reacting the acidic form of such a compound of Formula I        with a base which affords a pharmaceutically acceptable cation;        or for a zwitterionic compound of Formula I, neutralizing the        acid-addition salt form or base-addition salt form of such a        compound of Formula I; or by any other conventional procedure.

The present invention also provides for compounds of Formula I, whereinX is CH², R¹⁰ is fluoro, and the other variables are as defined above.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the invention have been found to be useful prodrugs ofcompounds that are selective agonists of metabotropic glutamatereceptors, and are therefore useful in the treatment of diseases of thecentral nervous system such as neurological diseases, for exampleneurodegenerative diseases, and as antipsychotic, anxiolytic,drug-withdrawal, antidepressant, anticonvulsant, analgesic andanti-emetic agents.

It will be appreciated that the compounds of Formula I contain at leastfour asymmetric carbon atoms, three being in the cyclopropane ring andone being at the α-carbon of the amino acid group. Accordingly, thecompounds of the invention may exist in and be isolated inenantiomerically pure form, in racemic form, or in a diastereoisomericmixture.

The amino acid moiety preferably has the natural amino acidconfiguration, i.e., the L-configuration relative to D-glycerolaldehyde.

The present invention includes pharmaceutically acceptable salts of acompound of Formula I. These salts can exist in conjunction with theacidic or basic portion of the molecule and can exist as acid addition,primary, secondary, tertiary, or quaternary ammonium, alkali metal, oralkaline earth metal salts. Generally, the acid addition salts may beprepared by the reaction of an acid with a compound of Formula I.Alternatively, acid addition salts may be prepared by reacting thepenultimate compound (protected intermediate) with appropriateequivalents of acid to produce the corresponding salt form, which, inturn, may be reacted to produce a compound of Formula I or other salts.The alkali metal and alkaline earth metal salts are generally preparedby the reaction of the hydroxide form of the desired metal salt with acompound of Formula I.

Some particular salts provide certain formulation advantages due totheir crystalline form. Non-crystalline amorphous forms of compounds maybe hygroscopic. Crystalline forms of pharmaceutical compounds aresometimes more desirable because they exhibit favorable solid-stateproperties.

Acids commonly employed to form such salts include inorganic acids, forexample hydrochloric, hydrobromic, nitric, sulphuric or phoshoric acids,or with organic acids, such as organic carboxylic acids, for example,glycollic, maleic, hydroxymaleic, fumaric, malic, tartaric, citric,salicyclic, o-acetoxybenzoic, or organic sulphonic, 2-hydroxyethanesulphonic, toluene-p-sulphonic, methane-sulfonic,naphthalene-2-sulphonic, benzene sulfonic, or ethane sulfonic acid.

In addition to pharmaceutically acceptable salts, other salts areincluded in the invention. They may serve as intermediates in thepurification of compounds or in the preparation of otherpharmaceutically acceptable acid addition salts, or are useful foridentification, characterization, or purification.

In the present invention, compounds of Formula I include solvatesthereof. Particularly, compounds of Formula I include hydrates thereof.

Furthermore, the present invention contemplates prodrugs of fluorinatedcompounds as disclosed in International Application Nos. PCT/JP99/03984,PCT/JP99/00324, and PCT/JP01/05550. See International Publication Nos.WO/0012464, WO/9938839, and WO/0200605, respectively. For example, thepresent invention contemplates prodrugs of1S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid;1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid; and1S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid.

A variety of physiological functions have been shown to be subject toinfluence by excessive or inappropriate stimulation of excitatory aminoacid transmission. The Formula I compounds of the present invention arebelieved to have the ability to treat a variety of neurologicaldisorders in mammals associated with this condition, including acuteneurological disorder such as cerebral deficits subsequent to cardiacbypass surgery and grafting, stroke, cerebral ischemia, spinal cordtrauma, head trauma, perinatal hypoxia, cardiac arrest, and hypoglycemicneuronal damage. The Formula I compounds are believed to have theability to treat a variety of chronic neurological disorders, such asAlzheimer's disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, ocular damage and retinopathy, cognitivedisorders, and idiopathic and drug-induced Parkinson's. The presentinvention also provides methods for treating these disorders whichcomprises administering to a patient in need thereof an effective amountof a compound of Formula I or a pharmaceutically acceptable saltthereof.

The Formula I compounds of the present invention treat a variety ofother neurological disorders in patients that are associated withglutamate dysfunction, including muscular spasms, convulsions, migraineheadaches, urinary incontinence, pain, premenstrual dysphoric disorder(PDD), psychosis, (such as schizophrenia), drug tolerance, withdrawal,cessation, and craving (such as nicotine, opiates, cocaine,benzodiazepines, and ethanol), anxiety and related disorders, emesis,brain edema, chronic pain, and tardive dyskinesia. The Formula Icompounds are also useful as antidepressant and analgesic agents.Therefore, the present invention also provides methods for treatingthese disorders which comprise administering to a patient in needthereof an effective amount of a compound of Formula I, or apharmaceutically acceptable salt thereof.

The following definitions are to set forth the meaning and scope of thevarious terms used herein. The general terms used herein have theirusual meanings.

The term “affecting” refers to a Formula II compound acting as anagonist at an excitatory amino acid receptor. The term “excitatory aminoacid receptor” refers to a metabotropic glutamate receptor, a receptorthat is coupled to cellular effectors via GTP-binding proteins. The term“cAMP-linked metabotropic glutamate receptor” refers to a metabotropicreceptor that is coupled to inhibition of adenylate cyclase activity.

The term “neurological disorder” refers to both acute and chronicneurodegenerative conditions, including cerebral deficits subsequent tocardiac bypass surgery and grafting, cerebral ischemia (for examplestroke resulting from cardiac arrest), spinal cord trauma, head trauma,Alzheimer's Disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, perinatal hypoxia, hypoglycemic neuronal damage,ocular damage and retinopathy, cognitive disorders, idiopathic anddrug-induced Parkinson's Disease. This term also includes otherneurological conditions that are caused by glutamate dysfunction,including muscular spasms, migraine headaches, urinary incontinence,drug tolerance, withdrawal, cessation, and craving (i.e. opiates,benzodiazepines, nicotine, cocaine, or ethanol), smoking cessation,emesis, brain edema, chronic pain, sleep disorders, convulsions,Tourette's syndrome, attention deficit disorder, and tardive dyskinesia.

The term “psychiatric disorder” refers to both acute and chronicpsychiatric conditions, including schizophrenia, anxiety and relateddisorders (e.g. panic attack and stress-related cardiovasculardisorders), depression, bipolar disorders, psychosis, obsessivecompulsive disorders, generalized anxiety disorder, acute stressdisorder, and panic disorder.

As used herein the term “effective amount” refers to the amount or doseof the compound, upon single or multiple dose administration to thepatient, which provides the desired effect in the patient underdiagnosis or treatment.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristics of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances. For example, a typicaldaily dose may contain from about 5 mg to about 300 mg of the activeingredient. The compounds can be administered by a variety of routesincluding oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, bucal, or intranasal routes. Alternatively, the compoundmay be administered by continuous infusion.

As used herein the term “patient” refers to a mammal, such as a mouse,guinea pig, rat, dog or human. It is understood that the preferredpatient is a human.

The term “treating” (or “treat”) as used herein includes its generallyaccepted meaning which encompasses prohibiting, preventing, restraining,and slowing, stopping, or reversing progression of a resultant symptom.As such, the methods of this invention encompass both therapeutic andprophylactic administration.

The general chemical terms used herein have their usual meanings. Forexample, the term “(1-6C) alkyl” means a straight or branched group.Examples of values for a (1-6C) alkyl group include (1-4C) alkyl such asmethyl, ethyl, propyl, isopropyl, butyl, and isobutyl. The term “(2-6C)alkenyl” includes (2-4C) alkenyl, such as allyl. The term “(2-6C)alkynyl” includes (2-4C) alkenyl, such as propynyl.

The term “optionally substituted,” as used in the terms “optionallysubstituted (1-6C) alkyl group,” “optionally substituted (2-6C) alkenylgroup,” and “optionally substituted (2-6C) alkynyl group,” hereinsignifies that one or more substituents may be present, preferably oneto three, said substituents being selected from atoms and groups which,when present in the compound of Formula I, do not prevent the compoundof Formula I from modulating metabotropic glutamate receptor function.

Examples of atoms and groups which may be present in an optionallysubstituted (1-6C) alkyl group, an optionally substituted (2-6C) alkenylgroup, or an optionally substituted (2-6C) alkynyl group are anoptionally substituted aromatic group, an optionally substitutedheteroaromatic group, a non-aromatic carbocyclic group, a non-aromaticheterocyclic group, a non-aromatic monocyclic carbocyclic group fusedwith one or two monocyclic aromatic or heteroaromatic groups and anon-aromatic monocyclic heterocyclic group fused with one or twomonocyclic aromatic or heteroaromatic groups.

The term “heteroaromatic group” includes an aromatic 5-6 membered ringcontaining from one to four heteroatoms selected from oxygen, sulfur andnitrogen, and an aromatic bicyclic group consisting of a 5-6 memberedring containing from one to four heteroatoms selected from oxygen,sulfur and nitrogen fused with a benzene ring or a 5-6 membered ringcontaining from one to four heteroatoms selected from oxygen, sulfur andnitrogen. Examples of heteroaromatic groups are furyl, thiophenyl,oxazolyl, isoxazolyl, thiazoyl, isothiazolyl, imidazolyl, pyrimidyl,benzofuryl, benzothiophenyl, benzimidazolyl, benzoxazolyl,benzothiazolyl and indolyl.

The term “aromatic group” includes phenyl and a polycyclic aromaticcarbocyclic ring such as naphthyl.

The term “optionally substituted,” as used in the terms “optionallysubstituted heteroaromatic group” and “optionally substituted aromaticgroup,” herein signifies that one or more substituents may be present,said substituents being selected from atoms and groups which, whenpresent in the compound of Formula I, do not prevent the compound ofFormula I from modulating metabotropic glutamate receptor function.

Examples of atoms and groups which may be present in an optionallysubstituted heteroaromatic or an optionally substituted aromatic groupare amino, hydroxy, nitro, halogeno, (1-6C) alkyl, (1-6C) alkoxy,(1-6C)alkylthio, carboxy, (1-6C) alkoxycarbonyl, carbamoyl, (1-6C)alkanoylamino, (1-6C)alkylsulphonyl, (1-6C) alkylsulphonylamino,optionally substituted phenyl, phenoxy, phenylthio, phenylsulphonyl,phenylsulphonylamino, toluenesulphonylamino, (1-6C)fluoroalkyl and(1-6C)fluoroalkoxy. Examples of particular values are amino, hydroxy,fluoro, chloro, bromo, iodo, methyl, methoxy, methylthio, carboxy,acetylamino, methanesulphonyl, nitro, acetyl, phenoxy, phenylthio,phenylsulphonyl, methanesulphonylamino and trifluoromethyl.

Examples of values for an optionally substituted aromatic group are1-naphthyl, 2-naphthyl, phenyl, 2-biphenyl, 3-biphenyl, 4-biphenyl,2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-fluorophenyl,3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl,pentafluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,2,4-dichlorophenyl, 3,4-dichlorophenyl, 3-chloro-4-fluorophenyl,3,5-dichlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl,2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methoxyphenyl,3-methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl,2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl,2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-fluoro-3-trifluoromethylphenyl,3-trifluoromethyl-4-fluorophenyl, 3-trifluoromethyl-5-fluorophenyl,2-fluoro-5-trifluoromethylphenyl, 2-phenoxyphenyl, 3-phenoxyphenyl,3-carboxyphenyl, and 4-carboxyphenyl.

The term “non-aromatic carbocyclic group” includes a monocyclic group,for example a (3-10C)cycloalkyl group, such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl orcyclodecyl, and a fused polycyclic group such as 1-adamantyl or2-adamantyl, 1-decalyl, 2-decalyl, 4a-decalyl, bicyclo[3,3,0]oct-1-yl,-2-yl or -3-yl, bicyclo[4,3,0]non-1-yl, -2-yl, -3-yl or -7-yl,bicyclo[5,3,0]dec-1-yl, -2-yl, -3-yl, -4-yl, -8-yl or -9-yl andbicyclo[3.3.1]non-1-yl, -2-yl, -3-yl or 9-yl.

The term “non-aromatic heterocyclic group” includes a 4 to 7 memberedring containing one or two heteroatoms selected from oxygen, sulphur andnitrogen, for example azetidin-1-yl or -2-yl, pyrrolidin-1-yl, -2-yl or-3-yl, piperidin-1-yl, -2-yl, -3-yl or 4-yl, hexahydroazepin-1-yl,-2-yl, -3-yl or -4-yl, oxetan-2-yl or -3-yl, tetrahydrofuran-2-yl or-3-yl, tetrahydropyran-2-yl, -3-yl or -4-yl, hexahydrooxepin-2-yl, -3-ylor -4-yl, thietan-2-yl or -3-yl, tetrahydrothiophen-2-yl or -3-yl,tetrahydrothiopyran-2-yl, -3-yl or -4-yl, hexahydrothiepin-2-yl, -3-ylor -4-yl, piperazin-1-yl or -2-yl, morpholin-1-yl, -2-yl or -3-yl,thiomorpholin-1-yl, -2-yl or -3-yl, tetrahydropyrimidin-1-yl, -2-yl,-4-yl or -5-yl, imidazolin-1-yl, -2-yl or -4-yl, imidazolidin-1-yl,-2-yl or -4-yl, oxazolin-2-yl, -3-yl, -4-yl or -5-yl, oxazolidin-2-yl,-3-yl, -4-yl or -5-yl, thiazolin-2-yl, -3-yl, -4-yl or -5-yl, orthiazolidin-2-yl, -3-yl, 4-yl or -5-yl.

The term “a non-aromatic monocyclic carbocyclic group fused with one ortwo monocyclic aromatic or heteroaromatic groups” includes a(3-10C)cycloalkyl group fused with a benzene ring or a an aromatic 5-6membered ring containing from one to four heteroatoms selected fromoxygen, sulfur and nitrogen, such as indanyl,1,2,3,4-tetrahydronaphth-1-yl or -2-yl, 5,6,7,8-tetrahydroquinolin-5-yl,-6-yl, -7-yl or 8-yl, 5,6,7,8-tetrahydroisoquinolin-5-yl, -6-yl, -7-ylor 8-yl, 4,5,6,7-tetrahydrobenzothiophen-4-yl, -5-yl, -6-yl or -7-yl,dibenzo[2,3,6,7]cycloheptan-1-yl or -4-yl,dibenzo[2,3,6,7]cyclohept-4-en-1-yl or -4-yl, or 9-fluorenyl.

The term “a non-aromatic monocyclic heterocyclic group fused with one ortwo monocyclic aromatic or heteroaromatic groups” includes a 4 to 7membered ring containing one or two heteroatoms selected from oxygen,sulphur and nitrogen, fused with a benzene ring or a an aromatic 5-6membered ring containing from one to four heteroatoms selected fromoxygen, sulfur and nitrogen, such as 2,3-dihydrobenzopyran-2-yl, -3-ylor -4-yl, xanthen-9-yl, 1,2,3,4-tetrahydroquinolin-1-yl, -2-yl, -3-yl or-4-yl, 9,10-dihydroacridin-9-yl or -10-yl,2,3-dihydrobenzothiopyran-2-yl, -3-yl or 4-yl, or dibenzothiopyran-4-yl.

The term “nitrogen-protecting group,” as used herein and as representedby “Pg^(N),” refers to those groups intended to protect or block thenitrogen group against undesirable reactions during syntheticprocedures. Choice of the suitable nitrogen-protecting group used willdepend upon the conditions that will be employed in subsequent reactionsteps wherein protection is required, as is well within the knowledge ofone of ordinary skill in the art. Commonly used nitrogen-protectinggroups are disclosed in T. W. Greene and P. G. M. Wuts, ProtectiveGroups In Organic Synthesis, 3^(rd) Ed. (John Wiley & Sons, New York(1999)). A preferred nitrogen-protecting group is tert-butoxycarbonyl.

The term “carboxy-protecting group,” as used herein and as representedby “Pg^(C),” refers to one of the ester derivatives of the carboxylicacid group commonly employed to block or protect the carboxylic acidgroup while reactions are carried out on other functional groups of thecompound. Particular values include, for example, methyl, ethyl,tert-butyl, benzyl, methoxymethyl, trimethylsilyl, and the like. Furtherexamples of such groups may be found in T. W. Greene and P. G. M. Wuts,Protecting Groups in Organic Synthesis, 3^(rd) Ed. (John Wiley & Sons,New York (1999)). Preferred carboxy-protecting group are methyl andethyl. The ester is decomposed by using a conventional procedure whichdoes not affect another portion of the molecule.

The term “hydroxyl protecting group” denotes a group understood by oneskilled in the organic chemical arts of the type described in Chapter 2of Greene and Wuts. Representative hydroxyl protecting groups include,for example, ether groups, substituted ethyl ether groups, isopropylether groups, phenyl and substituted phenyl ether groups, benzyl andsubstituted benzyl ether groups, alkylsilyl ether groups, esterprotecting groups, and the like. The species of hydroxyl protectinggroup employed is not critical so long as the derivatized hydroxyl groupis stable to the conditions of subsequent reaction(s) on other positionsof the intermediate molecule and can be selectively removed at theappropriate point without disrupting the remainder of the moleculeincluding any other hydroxyl protecting group(s).

The term “amino acyl” means an amino acyl derived from an amino acidselected from the group consisting of natural and unnatural amino acidsas defined herein. The natural amino acids may be neutral, positive ornegative depending on the substituents in the side chain. “Neutral aminoacid” means an amino acid containing uncharged side chain substituents.Exemplary neutral amino acids include alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, methionine, glycine,serine, threonine, cysteine, glutamine, and asparagine. “Positive aminoacid” means an amino acid in which the side chain substituents arepositively charged at physiological pH. Exemplary positive amino acidsinclude lysine, arginine and histidine. “Negative amino acid” means anamino acid in which the side chain substituents bear a net negativecharge at physiological pH. Exemplary negative amino acids includeaspartic acid and glutamic acid. Preferred amino acids are α-aminoacids. The most preferred amino acids are α-amino acids having Lstereochemistry at the α-carbon. Exemplary natural α-amino acids arevaline, isoleucine, proline, phenylalanine, tryptophan, methionine,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,lysine, arginine, histidine, aspartic acid and glutamic acid.

“Unnatural amino acid” means an amino acid for which there is no nucleicacid codon. Examples of unnatural amino acids include, for example, theD-isomers of the natural α-amino acids as indicated above; Aib(aminobutyric acid), βAib (3-aminoisobutyric acid), Nva (norvaline),β-Ala, Aad (2-aminoadipic acid), βAad (3-aminoadipic acid), Abu(2-aminobutyric acid), Gaba (γ-aminobutyric acid), Acp (6-aminocaproicacid), Dbu (2,4-diaminobutryic acid), α-aminopimelic acid, TMSA(trimethylsilyl-Ala), alle (allo-isoleucine), Nle (norleucine),tert-Leu, Cit (citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr(2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala),hydroxyproline, Sar (sarcosine), O-methyl tyrosine, phenyl glycine andthe like; cyclic amino acids; N^(α)-alkylated amino acids whereN^(α)-alkylated amino acid is N^(α)-(1-10C)alkyl amino acid such asMeGly (N^(α)-methylglycine), EtGly (N^(α)-ethylglycine) and EtAsn(N-ethylasparagine) and amino acids in which the α-carbon bears twoside-chain substituents. Exemplary unnatural α-amino acids includeD-alanine, D-leucine and phenylglycine. The names of natural andunnatural amino acids and residues thereof used herein follow the namingconventions suggested by the IUPAC-IUB Joint Commission on BiochemicalNomenclature (JCBN) as set out in “Nomenclature and Symbolism for AminoAcids and Peptides (Recommendations, 1983)” European Journal ofBiochemistry, 138, 9-37 (1984). To the extent that the names andabbreviations of amino acids and residues thereof employed in thisspecification and appended claims differ from those noted, differingnames and abbreviations will be made clear.

Although all of the compounds of Formula I are useful active mGluR2receptor agonists, certain compounds are preferred. The followingparagraphs define preferred classes.

-   -   A) Q is glycyl, alanyl, valyl, leucyl, isoleucyl, prolyl,        phenylalanyl, tyrosyl, tryptophyl, methionyl, lysyl, or serinyl.    -   B) Q is alanyl.    -   C) Q is methionyl.    -   D) p is 1.    -   E) p is 2.    -   F) X is SO₂.    -   G) X is CR³R⁴.    -   H) R³ is fluoro and R⁴ is hydrogen.    -   I) R³ is hydroxy and R⁴ is hydrogen.    -   J) R³ and R⁴ together represent ═O.    -   K) R¹⁰ is hydrogen.    -   L) R¹⁰ is fluoro.    -   M) R¹¹ is hydrogen.    -   N) The compound is a free base.    -   O) The compound is a salt.    -   P) The compound is the hydrochloride salt.    -   Q) The compound is the mesylate salt.    -   R) The compound is the esylate salt.    -   S) The compound is the tosylate salt.

The preceding paragraphs may be combined to define additional preferredclasses of compounds.

The compounds of Formula I are useful for the treatment of disorders ofmammals, and the preferred mammal is a human.

The compounds of the present invention can be prepared by a variety ofprocedures, some of which are illustrated in the schemes below. Theparticular order of steps required to produce the compounds of Formula Iis dependent upon the particular compound being synthesized, thestarting compound, and the relative lability of the substitutedmoieties. Some substituents may have been eliminated in the followingschemes for the sake of clarity, and are not intended to limit theteaching of the schemes in any way. As one of ordinary skill in the artwill appreciate, substituents R¹⁵ and R¹⁶ represent the appropriate sidechain to form the desired amino acyl.

If not commercially available, the necessary starting materials for thefollowing schemes may be made by procedures which are selected fromstandard techniques of organic and heterocyclic chemistry, techniqueswhich analogous to the syntheses of known, structurally similarcompounds, and the procedures described in the preparations andexamples, including novel procedures.

Compounds of Formula I are converted via enzymatic or hydrolytic processin vivo to form compounds of Formula II, as shown in Scheme 1 above. Inparticular, a crystalline form of a compound of Formula I may beprepared according to the route outlined in Scheme 2 below.

The hydrolysis of the diester protected peptidyl compound of formula(iii) with a suitable base such as lithium hydroxide or sodium hydroxidein a suitable solvent, such as THF or THF/water, affords the di-acidprotected peptidyl compound of formula (iv). A compound of formula (iv)may be deprotected with a suitable acid in a suitable solvent. Suchconditions may produce the corresponding acid salt of the di-acidpeptidyl compound, depicted in Formula I salt, as an amorphous solid or,directly, a crystalline solid, wherein X″ represents the correspondinganion. In the case of an amorphous solid, subsequent crystallization mayoccur from suitable solvents. Carboxylate salts may be formed by theintroduction of a cationic species by a reagent such as sodium acetate.Finally, the zwitterionic compound may be afforded by treatment of thecrystalline salt compound with an appropriate base.

For example, a di-acid protected peptidyl compound of formula (iv) whentreated hydrogen chloride gas in suitable solvent provides thedeprotected hydrochloride salt as an amorphous solid. The amorphoushydrochloride compound may then be crystallized from acetone and waterto afford the crystalline hydrochloride salt compound. In the case of acrystalline solid which is formed directly, filtration of the reactionmixture may afford the crystalline salt. The zwitterionic compound isafforded by treatment of the crystalline hydrochloride salt compoundwith sodium hydroxide; alternatively, treatment of the mesylate saltcompound or the tosylate salt compound with sodium hydroxide will alsoafford the zwitterionic compound. It will be appreciated by one ofordinary skill in the art that a compound of Formula I may be preparedin one procedure where the indicated intermediates are not isolated.

The di-ester of formula (ii) is acylated with a compound of Formula IIIusing a suitable coupling agent to afford a di-ester protected peptidylcompound of formula (iii). Alternatively, this transformation could beachieved using the acid chloride of a compound of Formula III.

Suitable peptide coupling reagents include dicyclohexylcarbodiimide(DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), isobutylchloroformate, diphenyl chlorophosphate,2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),bis(2-oxo-3-oxazolidinyl)phosphinic chloride, andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.

In Scheme 4 above, a compound of Formula II, a di-acid, is treated witha suitable carboxy-protecting agent, such as catalytic hydrochloric acidor thionyl chloride and methanol or ethanol, affording the correspondingdi-ester of formula (ii). Alternatively, a compound of Formula II firstmay be treated with a nitrogen-protecting agent such as BOC₂O to afforda nitrogen-protected compound of formula (i). Next, a compound offormula (i) may be treated with a carboxy-protecting agent such asmethyl iodide in the presence of a base such as potassium carbonate,followed then by an nitrogen deprotecting agent such as hydrochloricacid or trifluoroacetic acid to afford a compound of formula (ii).

Additionally, one of ordinary skill in the art would recognize thatdepending on X, an appropriate protecting agent may be necessary. Forexample, if X represents CR³R⁴, R³ represents hydroxy, and R⁴ representshydrogen, then one of ordinary skill in the art would appreciate that asuitable hydroxyl protecting group may be necessary before proceedingwith any of the above schemes.

Compounds of Formula II are known in the art. For example, preparationsof these compounds may be found in U.S. Pat. Nos. 5,688,826 (the '826patent) and 5,958,960 (the '960 patent).

Various improvements upon the synthetic route to compounds of Formula IIhave been made over the processes previously disclosed. The improvementsinvolve sulfur and alcohol oxidation, as well as optical resolution ofvarious intermediates as described below.

The first improvement relates to the conversion described in the '826patent at column 8, lines 22-34, and column 7, beginning at column 33(Formula V), involving oxidation of a compound of Formula VII of the'826 patent

to form a compound of Formula V of the '826 patent

It has been discovered that the sulfur trioxide-pyridine complex ortrifluoroacetic anhydride in conjunction with DMSO are preferred of themany oxidation methods known in the art.

Second, with respect to the resolution of a compound of Formula III ofthe '826 patent

in which R² represents a carboxyl group, referred to in column 8, atlines 3-7, and column 6, beginning at line 1 (Formula III), it has beendiscovered that (R)-α-methylbenzylamine and quinine are preferred.(R)-α-methylbenzylamine is particularly preferred.

Further, it has been discovered that when oxidizing the sulfide of acompound of Formula III of the '826 patent where X is sulfur to form acompound of Formula III of the '826 patent where X is sulfonyl, asreferred to in the '826 patent at column 8, lines 39-53, that a basicaqueous system and hydrogen peroxide used in combination with a catalystare preferred.

The following Examples further illustrate the compounds of the presentinvention and the methods for their synthesis. The Examples are notintended to be limiting to the scope of the invention in any respect,and should not be so construed. All experiments are run under a positivepressure of dry nitrogen or argon. All solvents and reagents arepurchased from commercial sources and used as received, unless otherwiseindicated. Dry tetrahydrofuran (THF) may be obtained by distillationfrom sodium or sodium benzophenone ketyl prior to use. Proton nuclearmagnetic resonance (¹H NMR) spectra are obtained on a Bruker Avance IIbay-500 at 500 MHz, a Bruker Avance I bay-200 at 200 MHz, or a VarianInova/Varian 300/Varian 400 at 500 MHz. Electrospray mass spectroscopy(ESI) is performed on a Agilent MSD/B instrument usingacetonitrile/aqueous ammonium acetate as the mobile phase. Free atombombardment mass spectroscopy (FABMS) is performed on a VG ZAB-2SEinstrument. Field desorption mass spectroscopy (FDMS) is performed usingeither a VG 70SE or a Varian MAT 731 instrument. Optical rotations aremeasured with a Perkin-Elmer 241 polarimeter. Chromatographic separationon a Waters Prep 500 LC is generally carried out using a linear gradientof the solvents indicated in the text. The reactions are generallymonitored for completion using thin layer chromatography (TLC). Thinlayer chromatography is performed using E. Merck Kieselgel 60 F₂₅₄plates, 5 cm×10 cm, 0.25 mm thickness. Spots are detected using acombination of UV and chemical detection (plates dipped in a cericammonium molybdate solution [75 g of ammonium molybdate and 4 g ofcerium (IV) sulfate in 500 mL of 10% aqueous sulfuric acid] and thenheated on a hot plate). Flash chromatography is performed as describedby Still, et al. Still, Kahn, and Mitra, J. Org. Chem., 43, 2923 (1978).Elemental analyses for carbon, hydrogen, and nitrogen are determined ona Control Equipment Corporation 440 Elemental Analyzer or are performedby the Universidad Complutense Analytical Centre (Facultad de Farmacia,Madrid, Spain). Melting points are determined in open glass capillarieson a Gallenkamp hot air bath melting point apparatus or a Büchi meltingpoint apparatus, and are uncorrected.

The abbreviations, symbols and terms used in the examples have thefollowing meanings.

-   -   Ac=acetyl    -   Anal.=elemental analysis    -   ATR=attenuated total internal reflection    -   Bn or Bzl=benzyl    -   Bu=butyl    -   BOC=t-butoxycarbonyl    -   calcd=calculated    -   D₂O=deuterium oxide    -   DCC=dicyclohexylcarbodiimide    -   DCM=1,2-dichloromethane    -   DIBAL-H=diisobutyl aluminum hydride    -   DMAP=4-dimethylaminopyridine    -   DMF=dimethylformamide    -   DMSO=dimethylsulfoxide    -   DSC=differential scanning calorimetry    -   EDC=N-ethyl-N′N′-dimethylaminopropyl carbodiimide hydrochloride    -   ES=Electrospray    -   Et=ethyl    -   EtOH=ethanol    -   FAB=Fast Atom Bombardment (Mass Spectrascopy)    -   FDMS=field desorption mass spectrum    -   FTIR=Fourier transform infrared spectrometry    -   HOAt=1-hydroxy-7-azabenzotriazole    -   HOBt=1-hydroxybenzotriazole    -   HPLC=High Performance Liquid Chromatography    -   HRMS=high resolution mass spectrum    -   i-PrOH=isopropanol    -   IR=Infrared Spectrum    -   L=liter    -   Me=methyl    -   MeOH=methanol    -   MPLC=Medium Pressure Liquid Chromatography    -   Mp=melting point    -   MTBE=t-butyl methyl ether    -   NBS=N-bromosuccinimide    -   NMR=Nuclear Magnetic Resonance    -   PC-TLC=preparative centrifugal thin layer chromatography    -   Ph=phenyl    -   p.o.=oral administration    -   i-Pr=isopropyl    -   Rochelle's Salt=potassium sodium tartrate    -   rt=room temperature    -   SM=starting material    -   TBS=tert-butyldimethylsilyl    -   TEA=triethylamine    -   Temp.=temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography    -   t-BOC=tert-butoxycarbonyl

General Procedure A EDC Coupling Between amines and N-BOC-(L)-aminoacids

Suspend the starting amino dialkyl ester (a compound of formula ii,Scheme 3) (1.0 equiv.) in dry dichloromethane under nitrogen.Sequentially add the corresponding N-Boc-(L)-aminoacid (1.5-2.0 equiv),EDC (1.5-2.0 equiv), HOBt (1.5-2.0 equiv), and dimethylaminopyridine(DMAP, 0.1-0.2 equiv). Stir the reaction mixture at room temperatureuntil judged complete by TLC unless otherwise noted. Dilute the reactionmixture with ethyl acetate and wash sequentially with saturated aqueousNaBCO₃ and/or aqueous NaHSO₄, and brine. After drying over sodiumsulfate and evaporation in vacuo purify the crude residue (a compound offormula iii) by silica gel chromatography using the appropriate eluent(typically ethyl acetate/hexanes).

General Procedure B Anhydride Coupling Between amine andN-BOC-(L)-aminoacid isobutyl anhydrides

To a solution of the corresponding N-Boc-(L)-aminoacid (1.5 equiv) indry dichloromethane (10 mL) at −20° C. under nitrogen add N-methylmorpholine (NMM, 1.5 equiv, in 1 mL CH₂Cl₂) followed by dropwiseaddition of iso-butyl chloroformate (IBCF, 1.5 equiv, in 5 mL CH₂Cl₂) ata rate so the internal reaction temperature does not exceed −15° C. Stirthe resulting reaction mixture at −20° C. for 30 minutes then add a −20°C. solution of(1S,2S,4S,5R,6R)-2-(2′-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (1.0 equiv) in dichloromethane (10 mL)at a rate so the internal reaction temperature does not exceed −15° C.Upon complete addition, remove the cooling bath and stir the reactionmixture at room temperature until judged complete by TLC. Dilute thereaction mixture with ethyl acetate and sequentially wash with saturatedaqueous NaHCO₃, aqueous NaHSO₄, and brine. After drying over magnesiumsulfate and evaporation in vacuo purify the crude residue by silica gelchromatography using the appropriate eluent (typically hexanes/ethylacetate).

General Procedure C

Sequential N-Boc and ester Protecting Group Removal

Stir the corresponding N-Boc diester peptide derivative (a compound offormula iii, Scheme 2) (1.0 equiv) in a 1:1 mixture of THF/2.5 N LiOH(10-20 equiv) at room temperature for up to 4 hours. Dilute the reactionwith H₂O and wash with ethyl acetate. Discard the organic layer. Adjustthe aqueous phase to pH 2 with 1 N HCl (NaCl added to aqueous phase toenhance extractability as needed) and exhaustively extract the N-bocdicarboxylic acid product (a compound of formula iv) with ethyl acetate.Combine all organics, wash with brine, dry over MgSO₄, and concentrateto dryness in vacuo to afford the desired carboxylate product as a foamysolid. Dissolve in ethyl acetate and chill to 0° C. Purge the reactionmixture with anhydrous HCl gas until saturated with HCl. Stir theresulting reaction mixture at 0° C. for up to 4 hours. Isolate the fullydeprotected peptide derivative (a compound of Formula I) as itshydrochloride salt by filtration under N₂ or by concentration of thereaction mixture to dryness followed by trituration with ethyl acetateor Et₂O and concentration to a white powder. Optionally, to removeresidual solvent and excess HCl, reconstitution of the products in H₂O,freezing, and subsequent lyophilization afford the desired hydrochlorideproducts.

Preparation 1(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester

To a slurry of(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ^(o)-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (10 g, 42.5 mmol, U.S. Pat. No. 5,688,826) in 100 mL of 2B ethanolat room temperature is added thionyl chloride (15.5 mL, 212.6 mmol)dropwise over 20 minutes followed by rinsing with 40 mL of ethanol. Heatthe slurry to reflux and stir overnight. Analysis via 500 MHz ¹H NMR(CD₃OD) of a concentrated aliquot reveals complete consumption ofstarting material and intermediate monoester. Allow the resultantsolution to cool to room temperature, then concentrate to a gelatinousresidue. Add EtOAc (50 mL) to the gelatin that was further concentratedto a solid, then dilute with another 94 mL of EtOAc. Add 15% aqueoussodium carbonate (70 mL) slowly to the mixture with swirling by hand togradually afford dissolution, giving a final pH of 7.95. Filter theresulting sodium carbonate precipitation before extracting the layers.Back extract the aqueous layer with EtOAc (2×100 mL). Wash the combinedorganic extracts with brine (1×100 mL), dry (MgSO₄), filter, andconcentrate in vacuo to provide a faint yellow oil that solidified togive the title compound as an off-white solid (11.71 g, 95% yield).

Recrystallization

A mixture of the title compound (200 mg) in EtOAc (800 ΔL) is heated to56° C. at which time dissolution occurs. After stirring for 15 minutesat 56° C., heptane (1 mL) is added dropwise to the solution. The heat isturned off. Allow the solution to cool to 52° C. at which timeprecipitation occurs. Upon cooling and further dilution with heptane(600 μL), the slurry forms. Stir the resultant slurry at roomtemperature for 1 hour before filtering, washing with heptane (2×500μL), and drying at 45° C. overnight to give 145 mg (73% recovery) of thetitle compound as a white solid.

mp 80-83° C.

[α]²⁵ _(D) −57.7° (c 1.04, CH₃OH).

500 MHz ¹H NMR (CD₃Cl₃) δ 4.31 (q, 2H, J=7.0 Hz), 4.20 (m, 2H), 3.78 (d,1H, J=15.0 Hz), 3.36 (dd, 1H, J=4.0, 7.0 Hz), 2.93 (dd, 1H, J=4.0, 7.0Hz), 2.81 (d, 1H, J=15.0 Hz), 2.46 (t, 1H, J=4.0), 1.34 (t, 3H, J=7.0),1.30 (t, 3H, J=7.0).

¹³C NMR (125 MHz, CD₃Cl₃) δ 171.68, 168.57, 63.26, 62.42, 59.96, 56.06,43.78, 32.25, 22.49, 14.31, 14.25.

FTIR (ATR) 3364.15 (s), 1725.95 (s), 1304.91 (s), 1259.24 (s), 1200.84(s), 1104.91 (s), 1022.99 (s), 896.45 (s), 851.21 (s) cm⁻¹.

Anal. Cald for C₁₁H₁₇NO₆S: C, 45.35; H, 5.88; N, 4.81. Found: C, 45.02;H, 5.75; N, 4.82.

Preparation 2 (1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester

To a solution of N-Boc-L-alanine (43.52 g, 230 mmol) and N-methylmorpholine (25.5 mL, 232 mmol) in 457 mL of methylene chloride at −30°C. under nitrogen add iso-butyl chloroformate (30.4 mL, 234 mmol)dropwise over 10 minutes. Stir the resultant thin slurry at −25 to −30°C. for 30 min at which time a solution of(1R,4S,5S,6S)-4-amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester (63.90 g, 219 mmol, Preparation 1) in 213 mL ofmethylene chloride is added over 25 minutes such that the reactiontemperature does not exceed −25° C. Upon completion of the addition,remove the cooling bath and allow to stir at ambient temperature for 60minutes at which time the reaction temperature reached 19° C. and thecolor became faint orange. Treat the reaction with 350 mL of 1 N HCl andseparate the layers. Wash the organic layer with saturated aqueousNaHCO₃ (1×350 mL) and brine (1×350 mL), dry (Na₂SO₄), filter, andconcentrate in vacuo to a white foam (105.2 g, 104%).

¹H NMR (300 MHz, CDCl₃) δ: 7.62 (brs, 1H), 4.90 (brd, 1H, J=7.1 Hz),4.34-4.10 (m, 6H), 3.39 (ddd, 1H, J=7.2, 3.9, 1.0 Hz), 3.00 (dd, 1H,J=7.1, 3.9 Hz), 2.90 (brd, 1H, J=14.9 Hz), 2.43 (t, 1H, J=4.1 Hz), 1.46(s, 9H), 1.31 (m, 9H).

¹³C NMR (75 MHz, CDCl₃) δ: 173.0, 168.6, 167.6, 80.9, 76.5, 63.3, 62.3,59.9, 55.7, 42.8, 31.5, 28.2, 22.7, 16.6, 14.0, 13.9. MS (ES) m/z 461.0[M−H]⁻.

Preparation 3(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid

To a solution of(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester (181.4 g, 392 mmol theoretical, Preparation 2) in 292mL of THF at room temperature add 490 mL (980 mmol) of 2N sodiumhydroxide. Allow the biphasic mixture to stir vigorously at roomtemperature for 1.25 hours at which time the reaction is homogeneous.Dilute the mixture with 490 mL of ethyl acetate and separate the layers.Dilute the aqueous layer with 490 mL of ethyl acetate, and lower the pHof the mixture to 1.5 with concentrated HCl. Separate the layers andback-extract the aqueous layer with 245 mL of ethyl acetate. Dry thecombined organic layers (Na₂SO₄), filter, and concentrate to provide167.9 g (105%) of the title compound as a white foam. This material wasused without characterization in Examples 1 and 2.

Preparation 4(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Add thionyl chloride (6.2 mL, 85.0 mmol) dropwise to a rapidly stirredsuspension of(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (10.0 g, 42.5 mmol, U.S. Pat. No. 5,688,826) in MeOH (170 mL, 5°C.). Upon complete addition, allow the reaction mixture to warm slowlyto room temperature, then warm under reflux for 48 h. Remove thevolatiles under reduced pressure, and partition the residue between asaturated solution of NaHCO₃ (200 mL) and ethyl acetate (400 mL).Separate the layers and extract the aqueous one with ethyl acetate(2×400 mL each time). Dry the combined organic layers over K₂CO₃, andconcentrate under reduced pressure to afford 8.10 g (30.8 mmol) of thetitle compound in 72% yield.

[α]_(D) ²³=−84 (c=0.5, MeOH).

Anal Calcd for C₉H₁₃NO₆S: C, 41.06; H, 4.98; N, 5.32. Found: C, 40.94;H, 4.93; N, 5.30.

MS (ES) m/z 264.0 [M+H]⁺.

Preparation 5(1R,4S,5S,6S)-4-(2′S-tert-Butoxycarbonylamino-3′-phenyl-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Phenylalanine and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux reaction mixture overnight.Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanes to 100%ethyl acetate). Yield 0.85 g (88%, 1.67 mmol) of a white foam.

[α]_(D) ²³=−35.2° (c=0.45, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.43 (9H, s), 2.38-2.40 (1H, m), 2.86 (1H, d,J=15.0 Hz), 2.91 (1H, dd, J=4.4, 7.3 Hz), 3.04 (2H, d, J=7.3 Hz),3.35-3.39 (1H, m), 3.77 (3H, s), 3.84 (3H, s), 4.11 (1H, d, J=14.3),4.30 (1H, app. q, J=7.3), 4.96 (1H, bd, J=6.6 Hz), 6.96 (1H, bs),7.22-7.36 (5H, m).

Anal Calcd for C₂₃H₃₀N₂O₉S.0.1H₂O: C, 53.92; H, 5.94; N, 5.47. Found: C,53.62; H, 5.90; N, 5.28.

MS (ES) m/z 509.16 [M−H]⁻; 411.2 [M−Boc]⁺.

Preparation 6(1R,4S,5S,6S)-4-(2′S-tert-Butoxycarbonylamino-3′S-methyl-pentanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Isoleucine and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux reaction mixture overnight.Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanes to 100%ethyl acetate). Yield 0.75 g (83%, 1.57 mmol) of a white foam.

[α]_(D) ²³=−32.65° (c=0.49, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 0.91 (3H, t, J=7.3 Hz), 0.93 (3H, d, J=6.6Hz), 1.10-1.18 (1H, m), 1.46 (9H, s), 1.42-1.52 (1H, m), 1.81-1.86 (1H,bm), 2.51 (1H, t, J=4.0 Hz), 2.95 (1H, d, J=15.0 Hz), 3.06 (1H, dd,J=4.4, 7.3 Hz), 3.43 (3H, dd, J=3.7, 7.0 Hz), 3.78 (3H, s), 3.85 (3H,s), 3.82-3.90 (1H, m), 4.20 (1H, d, J=14.7 Hz), 4.94 (1H, d, J=8.4 Hz),7.19 (1H, bs).

Anal Calcd for C₂₀H₃₂N₂O₉S: C, 50.41; H, 6.77; N, 5.88. Found: C, 50.32;H, 6.92; N, 5.76.

MS (ES) m/z 475.1 [M−H]⁻.

Preparation 7(1R,4S,5S,6S)-4-(2′S-tert-Butoxycarbonylamino-3′-methyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Valine and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux reaction mixture overnight.Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanes to 100%ethyl acetate). Yield 0.41 g (47%, 0.89 mmol) of a white foam.

[α]_(D) ²³=−35.36° (c=0.51, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 0.93 (3H, d, J=7.0 Hz), 0.96 (3H, d, J=6.6Hz), 1.46 (9H, s), 2.06-2.13 (1H, m), 2.50 (1H, t, J=4.0 Hz), 2.94 (1H,d, J=15.0 Hz), 3.04 (1H, dd, J=4.4, 7.3 Hz), 3.43 (1H, dd, J=3.3, 6.6),3.78 (3H, s), 3.80-3.86 (1H, m), 3.86 (3H, s), 4.24 (1H, d, J=15.0 Hz),4.94 (1H, d, J=8.1 Hz), 7.15 (1H, bs).

Anal Calcd for C₁₉H₃BN₂O₉S: C, 49.34; H, 6.54; N, 6.06. Found: C, 49.33;H, 6.44; N, 6.05.

MS (ES) m/z 461.2 [M−H]⁻

Preparation 8(1R,4S,5S,6S)-4-(2′S-tert-Butoxycarbonylamino-4′-methyl-pentanoylamino)-2,2-d2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylic acid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Leucine monohydrate and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux the reaction mixtureovernight. Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanesto 100% ethyl acetate). Yield 0.85 g (94%, 1.78 mmol) of a white foam.

[α]_(D) ²³=−46.15° (c=1.04, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 0.92 (3H, d, J=6.2 Hz), 0.95 (3H, d, J=6.6Hz), 1.47 (9H, s), 1.42-1.47 (1H, m), 1.63-1.67 (1H, m), 2.46 (1H, t,J=3.7 Hz), 2.87 (1H, d, J=15.0 Hz), 3.04 (1H, dd, J=4.4, 7.3 Hz), 3.41(1R, dd, J=3.7, 7.0), 3.78 (3H, s), 3.86 (3H, s), 4.00-4.05 (1H, m),4.20 (1H, d, J=15.0 Hz), 4.75 (1H, d, J=6.6 Hz), 7.43 (1H, bs).

Anal Calcd for C₂₀H₃₂N₂O₉S: C, 50.41; H, 6.77; N, 5.88. Found: C, 50.30;H, 6.82; N, 5.75.

MS (ES) m/z 475.2 [M−H]⁻.

Preparation 9(1R,4S,5S,6S)-4-(2′S,6′-bis-tert-butoxycarbonylamino-hexanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-Lys(BOC)-OH and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2%6-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux the reaction mixtureovernight. Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanesto 100% ethyl acetate). Yield 1.04 g (93%, 1.76 mmol) of a white foam.

[α]_(D) ²³=−32.0° (c=0.5, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.44 (9H, s), 1.46 (9H, s), 1.39-1.53 (3H, m),1.56-1.65 (1H, m), 1.77-1.84 (2H, m), 2.50 (1H, t, J=4.4 Hz), 2.98-3.20(4H, m), 3.42 (1H, dd, J=3.7, 7.0 Hz), 3.76 (3H, s), 3.86 (3H, s), 4.01(1H, dd, J=7.7, 13.2 Hz), 4.09-4.19 (1H, m), 4.71 (1H, t, J=7.3 Hz),5.13 (1H, bs), 7.59 (1H, bs).

Anal Calcd for C₂₅H₄₁N₃O₁₁S: C, 50.75; H, 6.98; N, 7.10. Found: C,50.36; H, 6.99; N, 6.87.

MS (ES) m/z 590.2 [M−H]⁻.

Preparation 10(1R,4S,5S,6S)-4-[2′S-tert-Butoxycarbonylamino-4′-(trityl-carbamoyl)-butyrylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Glutamine (Trt)-OH and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 4). Reflux the reaction mixtureovernight. Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanesto 100% ethyl acetate). Yield 0.53 g (48%, 0.72 mmol) of a white foam.

[α]_(D) ²³=−8.0° (c=0.50, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 1.42 (9H, s), 1.83-1.88 (1H, m), 2.03-2.18(1H, m), 2.16 (1H, t, J=4.0 Hz), 2.57-2.64 (1H, m), 2.60 (1H, d, J=15.0Hz), 2.64-2.80 (1H, m), 2.88 (1H, dd, J=4.4, 7.3 Hz), 3.26 (1H, dd,J=4.0, 7.0 Hz), 3.47 (3H, s), 3.76-3.90 (1H, m), 3.81 (3H, s), 4.05 (1H,d, J=15.0 Hz), 5.47 (1H, bs), 7.02 (1H, bs), 7.20-7.35 (15H, m), 8.68(1H, bs).

Anal Calcd for C₃₈H₄₃N₃O₁₀S: C, 62.20; H, 5.91; N, 5.73. Found: C,61.83; H, 6.09; N, 5.57.

MS (ES) m/z 731.9 [M−H]⁻;

-   -   HRMS calcd for C₃₈H₄₃N₃O₁₀S [M+Na]⁺, 756.2567. Found, 756.2585.

Preparation 11(1R,4S,5S,6S)-4-[1′-tert-Butoxycarbonyl-pyrrolidine-2′S-carbonyl)-amino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableBoc-(L)-proline (0.61 g, 2.9 mmol) and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.5 g, 1.9 mmol, Preparation 4). Purify usingPC-TLC (ethyl acetate/hexanes) to yield 0.87 gram (99.2%) of the titlecompound.

[α]_(D) ²³=−61.2 (c=0.49, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.51 (9H, s), 1.75-1.98 (3H, m), 2.35-2.5 (2H,m), 2.84 (1H, d, J=14.7 Hz), 2.9-3.03 (1H, m), 3.25-3.4 (1H, m), 3.3-4.1(2H, m), 3.76 (3H, s), 3.86 (3H, s), 4.14-4.31 (2H, m), 8.66 (1H, s).

MS (ES) m/z 459.2 [M−1]⁻.

Preparation 12(1R,4S,5S,6S)-4-(2′S-tert-Butoxycarbonylamino-4′-methylsulfanyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableBoc-(L)-Methionine (0.71 g, 2.9 mmol) and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2)₆-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.5 g, 1.9 mmol, Preparation 4). Purify usingPC-TLC (ethyl acetate/hexanes) to yield 0.85 gram (90.5%) of the titlecompound.

[α]_(D) ²³=−20.4 (c=0.49, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.46 (9H, s), 1.69 (1H, s), 1.8-2.05 (2H, m),1.9-2.0 (1H, m), 1.95-2.3 (3H, bs), 2.0-2.2 (1H, m), 2.4-2.8 (2H, m),2.48 (1H, t, J=4.0 Hz), 2.58 (1H, bs), 2.92 (1H, d, J=14.7 Hz), 3.01(1H, dd, J=4.4, 7.0 Hz), 3.42 (1H, dd, J=3.7, 7.3 Hz), 3.78 (3H, s),3.87 (3H, s), 4.22-4.24 (2H, m), 5.06 (1H, d, J=7.7 Hz), 7.27 (1H, s).

MS (ES) m/z 493.1 [M−1]⁻.

Preparation 13(1R,4S,5S,6S)-4-[2′S-tert-Butoxycarbonylamino-3′-(1-tert-butoxycarbonyl-1H-indol-3-yl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially availableBoc-(L)-Tryptophan(Boc) (1.1 g, 2.8 mmol) and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.5 g, 0.9 mmol, Preparation 4). Purify usingPC-TLC (ethyl acetate/hexanes) to yield 0.7 g (56.7%) of the titlecompound.

¹H NMR (300 MHz, CDCl₃) δ 1.44 (9H, s), 1.67 (9H, s), 2.37 (1H, bs),2.86 (1H, d, J=15.0 Hz), 2.88 (1H, t, J=4.4 Hz), 3.15 (2H, d, J=6.6 Hz),3.39 (1H, dd, J=3.7, 7.0 Hz), 3.73 (3H, s), 3.83 (3H, s), 4.18 (1H, d,J=14.7 Hz), 4.37-4.44 (1H, m), 5.01 (1H, bd, J=8.1 Hz), 7.11 (1H, bs),7.25-7.59 (4H, m), 8.14 (1H, bd, J=8.4 Hz).

[α]_(D) ²³=−19.6 (c=0.51, CHCl₃).

Anal. Calcd. For C₃₀H₃₉N₃O₁₁S.10C₄H₈O₂: C, 55.35; H, 6.42; N, 5.70.Found: C, 54.98; H, 6.09; N, 6.07.

HRMS calcd for C₃₀H₃₉N₃O₁₁Na₁S, 672.2203. Found, 672.2180.

Preparation 14(1R,4S,5S,6S)-4-[2′S-tert-Butoxycarbonylamino-3′-(4-tert-butoxycarbonyloxy-phenyl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using commercially available2S-tert-butoxycarbonylamino-3-(4-tert-butoxycarbonyloxy-phenyl)-propionicacid (1.1 g, 2.9 mmol) and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.5 g, 1.9 mmol, Preparation 4). Purify usingPC-TLC (ethyl acetate/hexanes) to yield 0.94 g (79.0%) of the titlecompound.

[α]_(D) ²³=+4 (c=1.00, CH₃OH).

¹H NMR (300 MHz, CDCl₃) δ 1.44 (9H, s), 1.56 (9H, s), 2.44 (1H, t, J=4.0Hz), 2.88 (1H, d, J=14.7 Hz), 2.98 (1H, dd, J=4.4, 7.3 Hz), 3.04 (2H, d,J=7.3 Hz), 3.38 (1H, dd, J=4.0, 7.3 Hz), 3.77 (3H, s), 3.83 (3H, s),4.11 (1H, d, J=14.3 Hz), 4.22-4.29 (1H, app q, J=7.3 Hz), 4.92 (1H, bd,J=7.7 Hz), 7.07 (1H, bs), 7.1-7.26 (4H, m).

HRMS calcd for C₂₈H₃₈N₂O₁₂SNa, 649.2043. Found, 649.2001.

Preparation 15(1R,4S,5S,6S)-4-(3′-Acetoxy-2′S-tert-butoxycarbonylamino-propionyl)amino-2,2-dioxo-2λ⁶-thiabicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester

Prepare according to General Procedure A using3-acetoxy-2S-(tert-butoxycarbonylamino)propionic acid (0.25 g, 1.0 mmol,Preparation 44) and(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.2 g, 0.8 mmol, Preparation 4) with the exceptionthat DMAP is not used. Purify using PC-TLC (ethyl acetate/hexanes) toyield 0.19 g (48.2%) of the title compound.

[α]_(D) ²³=−24 (c=1.0, CH₃OH).

¹H NMR (300 MHz, CDCl₃) δ 1.47 (9H, s), 2.10 (3H, s), 2.51 (1H, t, J=4.4Hz), 2.99-3.07(2H, m), 3.43 (1H, dd, J=4.0, 7.3 Hz), 3.77(3H, s), 3.86(3H, s), 4.14-4.40 (4H, m), 5.29 (1H, bd, J=7.3 Hz), 7.64 (1H, bs).

HRMS calcd for C₁₉H₂₈N₂O₁₁SNa, 515.1312. Found, 515.1305.

Preparation 16(1R,2S,4R,5R,6R-2-Amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

To a slurry of(1R,2S,4R,5R,6R)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (14.45 g, 71.12 mmol, U.S. Pat. No. 5,958,960) in 202 mL ofabsolute ethanol at room temperature add thionyl chloride (26 mL, 356mmol) dropwise over 20 minutes. Heat the slurry to reflux and allow tostir for 3 hours. Allow to cool to room temperature and stir overnight.Concentrate the resultant solution in vacuo to a residue then dilutewith 136 mL of ethyl acetate and treat with 306 mL of 10% aqueous sodiumcarbonate over 15 minutes with swirling by hand such that the final pHis 10. Separate the layers and wash the aqueous layer with ethyl acetate(1×136 mL). Wash the combined organic extracts with brine (1×136 mL),dry (MgSO₄), filter, and concentrate in vacuo to provide 17.07 g (93%)of the title compound as white solid.

[α]_(D) ²³=+20.37° (c=1.1, MeOH).

m.p.=64-660C.

¹H NMR (400 MHz, CDCl₃) δ 1.28 (3H, t, J=7.3 Hz), 1.31 (3H, t, J=6.8Hz), 1.34-1.45 (1H, m), 1.85 (2H, bs), 2.17-2.21 (2H, m), 2.32-2.34 (1H,m), 2.49 (1H, dd, J=7.8, 14.1 Hz), 4.24 (2H, dq, J=1.5, 7.3 Hz),5.33-5.52 (1H, m). Anal. calcd. for C₁₂H₁₈FNO₄: C, 55.59; H, 7.00; N,5.40. Found: C, 55.29; H, 6.75; N, 5.45.

MS (ES) m/z found 260.3 [M+H]+.

Preparation 17(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]amino-4-flurobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL ofmethylene chloride at −22° C. under nitrogen add N-methyl morpholine(22.44 mL, 204 mmol) followed by iso-butyl chloroformate (26.48 mL, 204mmol) dropwise over 15 min such that the reaction temperature does notexceed −18° C. Allow the resultant thin slurry to stir at −20° C. for 30minutes at which time a solution of(1R,2S,4R,5R,6R)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (49.46 g, 191 mmol, Preparation 16) in 247 mL ofmethylene chloride is added over 40 min such that the reactiontemperature does not exceed −16° C. Upon completion of the addition,remove the reaction from the cooling bath and allow to stir at ambienttemperature for 70 minutes at which time the reaction temperaturereached 15° C. and the color became faint orange. Treat the reactionwith 408 mL of 1 N HCl and stir for 5 minutes then separate the layers.Wash the organic layer with saturated aqueous sodium bicarbonate (1×408mL), dry (Na₂SO₄), filter, and concentrate in vacuo to a white foam(88.16 g).

Anal. calcd. For C₂₀H₃₁FN₂O₇.0.1CH₂Cl₂: C, 55.00; H, 7.16; N, 6.38.Found: C, 55.18; H, 7.18; N, 6.49.

MS (ES) m/z 431.3 [M+H]+, 331.2 [M+H-Boc]+.

Preparation 18(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid

To a solution of(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]-amino-4-flurobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (88.16 g, 191 mmol, Preparation 17) in 238 mL of THFat room temperature add 238 mL (477 mmol) of 2N sodium hydroxide. Allowthe biphasic mixture to stir vigorously at room temperature for 2.5hours at which time the reaction is homogeneous. Dilute the mixture with238 mL of t-butyl methyl ether followed by mixing and separation of thelayers. Further dilute the aqueous layer with 238 mL of water and filterto remove particulate matter. Treat the solution with concentrated HCl(42.9 mL, 515 mmol) over 30 minutes optionally followed by seeding withthe title compound and stirring for 1 hour. Filter the resultant slurry,wash with water (2×100 mL), and vacuum dry at 45° C. for 40 hours toprovide 72.2 g of the title compound as a white solid. Stir a portion ofthe solid (69.5 g) with 490 mL of acetone for 1 hour to produce a hazysolution; filter and wash with acetone (2×100 mL). Concentrate thefiltrate in vacuo to a white foam which is further dried in vauo at 45°C. for 16 hours to provide 61.8 g (86%, corrected for 12% wt/wt acetone)of the title compound. This material was used in Examples 14-18 withoutcharacterization.

Preparation 19(1S,2S,4S,5R,6R)-4-Acetyloxy-2-(tert-butoxycarbonyl)aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

To a solution of(1S,2S,4S,5R,6R)-2-(tert-butoxycarbonyl)amino-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (1.50 g, 4.20 mmol, U.S. Pat. No. 5,958,960),pyridine (0.365 mL, 4.62 mmol) and DMAP (0.513 g, 4.20 mmol) indichloromethane (40 mL) under nitrogen add acetic anhydride (0.514 mL,5.0 mmol). Stir at room temperature for 16 hours, dilute withdichloromethane, and pour into 10% aqueous citric acid solution (50 mL).Wash the organic layer with water (50 mL) and brine (50 mL). Dry overMgSO₄, filter and concentrate in vacuo, to produce the title compound asa white solid (1.295 g, 75%).

LCMS: m/z 400 [M+H]⁺ and m/z 300 [M+H—CO₂ ^(t)Bu]⁺ @ R_(T) 1.39 min.

Preparation 20(1S,2S,4S,5R,6R)-4-Acetyloxy-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Dissolve(1S,2S,4S,5R,6R)₄-acetyloxy-2-(tert-butoxycarbonyl)aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (1.25 g, 3.13 mmol, Preparation 19) in a solution of95% TFA in dichloromethane (60 mL) and stir under nitrogen at roomtemperature for 5 minutes. After this time remove theTFA/dichloromethane in vacuo. Dissolve the crude product in a suspensionof NaHCO₃ (1.00 g) in dichloromethane (50 mL) and stir for 30 minutes.Filter the suspension, wash with dichloromethane (3×25 mL), andconcentrate in vacuo to afford 916 mg (98%) of the product as a yellowoil.

LCMS: m/z 300 [M+H]⁺ @ R_(T) 0.92 min.

Preparation 21(1S,2S,4S,5R,6R)-4-Acetyloxy-2-[2′S-(tert-butoxy)carbonylaminopropionyl]-aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

To a suspension of(1S,2S,4S,5R,6R)-4-acetyloxy-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (0.80 g, 1.94 mmol, Preparation 20) and Boc-L-Ala(0.477 g, 2.52 mmol) in anhydrous dichloromethane (50 mL) under nitrogensequentially add EDC (0.519 g, 2.72 mmol), HOBt (0.314 g, 2.32 mmol),catalytic DMAP (0.024 g, 0.19 mmol), and triethylamine (1.08 mL, 7.76mmol). After stirring at room temperature for ca. 15 minutes, theinitial white suspension dissolves completely. Stir the reaction for 16hours, dilute with dichloromethane, and wash with saturated aqueousNaHCO₃ solution (50 mL), 1.0 N aqueous HCl solution (3×20 mL), andsaturated brine solution (40 mL). Dry the organic layer over MgSO₄ andfilter, and concentrate in vacuo. Purify the resulting crude amide bycolumn chromatography using a (4:1) mixture of ethyl acetate and hexanesas eluent to afford 638 mg (75%) of the product as a white solid.

¹H NMR (CD₃OD)*: δ 5.05 (1H, d, 5.7 Hz), 4.09 (2H, q, 7.3 Hz), 4.02 (2H,q, 7.3 Hz), 4.01-3.94 (1H, m), 2.65-2.59 (2H, m), 2.38 (1H, d, 14 Hz),2.10-2.03 (3H, m), 1.91 (3H, s), 1.78 (1H, dd, 5.9 Hz, 16 Hz), 1.59 (1H,br s), 1.34 (9H, s), 1.16 (6H, 2×t, 7.3 Hz); *N.B. Exchangeable protonsnot observed by NMR=2.

LCMS: m/z 471 [M+H]⁺ and m/z 371 [M+H—CO₂ ^(t)Bu]⁺ @ R_(T) 1.30 min.

R_(f) 0.50 (80% ethyl acetate: heptane).

Preparation 22(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester

Cool a solution of(1S,2S,4S,5R,6R)-2-tert-butoxycarbonylamino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (5.0 g, 14.0 mmol, U.S. Pat. No. 5,958,960) in ethylacetate to 0° C. Bubble anhydrous HCl (g) into the solution untilsaturated and stir for 0.5 hour. Allow the reaction to warm to roomtemperature and stir for 1 hour. Concentrate the reaction mixture andpartition between ethyl acetate and H₂O. Treat the aqueous layer withNaHCO₃ (aq) and extract with ethyl acetate. Dry the organics with K₂CO₃and concentrate to yield 2.2 g (59.7%) of white solids.

[α]_(D) ²³=−30.8 (c=0.52, CH₃OH).

¹H NMR (300 MHz, CDCl₃) δ 1.26 (3H, t, J=7.33 Hz), 1.36 (3H, t, 7.33Hz), 1.60 (1H, dd, J=5.87, 15.40 Hz), 1.64 (1H, t, J=2.93 Hz), 2.27 (2H,bs), 2.15-2.29 (3H, m), 3.93 (1H, bs), 4.13 (2H, q, J=6.97 Hz),4.27-4.36 (3H, m).

Anal. Calcd. For C₁₂H₁₉NO₅: C, 56.02; H, 7.44; N, 5.44. Found: C, 55.75;H, 7.36; N, 5.40.

MS (ES) m/z 258.1 [M+H]⁺.

Preparation 23(1S,2S,4S,5R,6R)-2-(2′-tert-Butoxycarbonylamino-acetylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-Glycine (458 mg, 2.62mmol, Aldrich) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (450 mg, 1.75 mmol, Preparation 22). Purify on 35 gof silica eluting with a gradient from 50/50 to 20/80 hexanes/ethylacetate. Yield: 560 mg (77%).

¹H NMR (400 MHz, CDCl₃) δ 1.28 (3H, t, J=7.3 Hz), 1.29 (3H, t, J=7.3Hz), 1.48 (9H, s), 1.57 (2H, m), 2.21 (1H, m), 2.41 (1H, dd, J=2.9, 5.8Hz), 2.81 (1H, d, J=15.6 Hz), 3.76 (1H, d, J=5.8 Hz), 4.10-4.18 (3H, m),4.26 (2H, q, J=7.3 Hz), 4.33 (1H, m), 5.10 (1H, bs), 6.86 (1H, bs).

Anal Calcd for C₁₉H₃₀N₂O₈: C, 55.06; H, 7.30; N, 6.76. Found: C, 55.24;H, 7.50; N, 6.76.

MS (ES) m/z 415.2 [M+H]⁺, 437.2 [M+Na]⁺.

Preparation 24(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-3′-methyl-butyrylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-Valine (569 mg,2.62 mmol, Sigma) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (450 mg, 1.75 mmol, Preparation 22). Purify the crudematerial on 35 g of silica; elute with a gradient from 70/30 to 20/80hexanes/ethyl acetate. Yield: 694 mg (87%) of a white foam.

¹H NMR (400 MHz, CDCl₃) δ 0.95 (3H, d, J=6.8 Hz), 1.00 (3H, d, J=6.8Hz), 1.29 (6H, t, J=7.3 Hz), 1.45 (9H, s), 1.52-1.60 (2H, m), 2.08 (1H,m), 2.20 (1H, m), 2.42 (1H, m), 2.81 (1H, d, J=115.6 Hz), 3.86 (1H, dd,J=6.3, 8.8 Hz), 4.14 (2H, q, J=7.3 Hz), 4.21-4.32 (4H, m), 5.00 (1H, d,J=8.3 Hz), 6.63 (1H, s).

Anal Calcd for C₂₂H₃₆N₂O₈: C, 57.88; H, 7.95; N, 6.14. Found: C, 57.87;H, 8.03; N, 6.12.

MS (ES) m/z 457.2 [M+H]⁺, 479.2 [M+Na]⁺.

Preparation 25(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-4′-methyl-pentanoylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-Leucine (606 mg,2.62 mmol, Chemlog) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl (450 mg, 1.75 mmol, Preparation 22). Purify the crudematerial on 35 g of silica; elute with a gradient from 70/30 to 20/80hexanes/ethyl acetate. Yield 689 mg (84%) of a white foam.

¹H NMR (400 MHz, CDCl₃) δ 0.96 (6H, m), 1.28 (3H, t, J=7.3 Hz), 1.29(3H, t, J=7.3 Hz), 1.46 (9H, s), 1.45-1.73 (5H, m), 2.20 (1H, m), 2.42(1H, m), 2.80 (1H, d, J=16.1 Hz), 4.07-4.35 (7H, m), 4.81 (1H, bd), 6.86(1H, bs).

Anal Calcd for C₂₃H₃₈N₂O₈.0.1H₂O: C, 58.48; H, 8.15; N, 5.93. Found: C,58.22; H, 7.94; N, 5.92.

MS (ES) m/z 471.2 [M+H]⁺, 493.2 [M+Na]⁺.

Preparation 26(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-3′S-methyl-pentanoylamino)₄-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-Isoleucine (606 mg,2.62 mmol, Aldrich) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (450 mg, 1.75 mmol, Preparation 22). Purify the crudematerial on 35 g of silica eluting with a gradient from 70/30 to 20/80hexanes/ethyl acetate. Yield: 731 mg (89%).

¹H NMR (400 MHz, CDCl₃) δ 0.92 (3H, t, J=7.3 Hz), 0.97 (3H, d, J=6.8Hz), 1.17 (1H,m), 1.29 (6H, t, J=6.8 Hz), 1.46 (9H, s), 1.53 (1H, dd,J=5.8, 15.6 Hz), 1.58 (1H, t, J=2.4 Hz), 1.74 (1H, m), 1.83 (1H, m),2.20 (1H, m), 2.43 (1H, m), 2.80 (1H, d, J=15.6 Hz), 3.88 (1H, dd,J=7.3, 8.8 Hz), 4.12-4.32 (6H, m), 4.98 (1H, d, J=7.3 Hz), 6.60 (1H, s).

Anal Calcd for C₂₃H₃₈N₂O₈.0.2H₂O: C, 58.25; H, 8.16; N, 5.91. Found: C,58.17; H, 8.11; N, 5.91.

MS (ES) m/z 471.2 [M+H]⁺, 493.2 [M+Na]⁺.

Preparation 27(1S,4S,5R,6R)-2-[2′-(2-tert-Butoxycarbonylamino-acetylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-Gly-Gly (474 mg, 2.04mmol) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (400 mg, 1.36 mmol, Preparation 22without basic work-up) with the following exceptions. No DMAP is used.Add one equivalent of triethylamine. Purify the crude material on 35 gof silica; elute with ethyl acetate. Yield: 517 mg (81%).

[α]_(D) ²³ =−18.18 (c=0.55, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 1.26 (3H, t, J=6.8 Hz), 1.27 (1H, t, J=7.3Hz), 1.28 (3H, t, J=6.8 Hz), 1.46 (9H, s), 1.59 (1H, t, J=3.4 Hz), 1.62(1H, dd, J=6.3, 16.1 Hz), 2.21 (1H, dd, J=2.9, 5.8 Hz), 2.46 (1H, dd,J=2.4, 5.8 Hz), 2.73 (1H, d, J=15.6 Hz), 3.78-3.91 (3H, m), 4.01-4.15(3H, m), 4.24 (2H, q, J=6.8 Hz), 4.32 (1H, d, J=5.8 Hz), 5.28 (1H, b),6.87 (1H, bt, J=4.9 Hz), 7.39 (1H, bs).

HRMS calcd for C₂₁H₃₄N₃O₉, 472.2295. Found, 472.2303.

HPLC: 16.755 min. Column: Symmetry C18, 3.5 um, 4.6×150 mm. λ=230 nM.Flow Rate: 1 mL/min. Gradient: 10% to 50% ACN/water containing 0.1% TFAover 25 min.

Preparation 28(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-Butoxycarbonylamino-propionylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-Ala-Gly (502 mg, 2.04mmol) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (400 mg, 1.36 mmol, Preparation 22without basic work-up) with the following exceptions. No DMAP is used.Add one equivalent of triethylamine. Purify the crude material on 35 gof silica eluting with ethyl acetate. Yield: 500 mg (76%).

[α]_(D) ²³=−31.37(c=0.55, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 1.27 (3H, t, J=6.8 Hz), 1.28 (3H, t, J=6.8Hz), 1.42 (3H, d, J=6.8 Hz), 1.45 (9H, s), 1.58 (1H, t, J=3.4 Hz), 1.64(1H, dd, J=5.8, 15.6 Hz), 2.23 (1H, dd, J=3.4, 5.8 Hz), 2.50 (1H, dd,J=2.9, 6.3 Hz), 2.69 (1H, d, J=15.6 Hz), ABq of doublets (2H,ν_(A)=3.87, ν_(B)=3.98 J_(AB)=17.1 Hz, J_(d)=6.3 Hz), 3.92 (1H, m),4.07-4.16 (3H, m), 4.24 (2H, q, J=7.3 Hz), 4.32 (1H, b), 5.08 (1H, b),6.84 (1H, bt, J=4.88 Hz), 7.14 (1H, bs).

HRMS calcd for C₂₂H₃₆N₃O₉, 486.2452. Found, 486.2451.

Preparation 29(1S,2S,4S,5R,6R)-2-(2′-tert-Butoxycarbonylamino-3′-phenyl-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to the General Procedure A using Boc-L-phenylalanine(772 mg, 2.91 mmoles) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (480 mg, 1.94 mmoles, Preparation 22). Concentratethe crude reaction mixture, dissolve in dichloromethane, flash through ashort silica gel plug with ethyl acetate/DCM (1:1), and concentratein-vacuo to give a yellow oil. Further purify the material by silica gelchromatography eluting with 30% ethyl acetate/hexanes to 80% ethylacetate/hexanes to afford 852 mg (87%) of the title compound.

[α]_(D) ²³=−23.66 (c=0.93, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.22-1.30 (6H, m), 1.35 (9H, s), 1.59 (1H, t,J=2.9 Hz), 1.70 (1H, dd, J=5.5, 15.4 Hz), 2.06 (1H, m), 2.40 (1H, d,J=15.4 Hz), 2.63 (1H, dd, J=2.9, 5.9 Hz), 2.76 (1H, dd, J=9.2, 13.9 Hz),4.08-4.33 (6H, m), 7.20-7.29 (5H, m).

MS found 505.0 [M+H]⁺.

HRMS calcd for C₂₆H₃₆N₂O₈, 505.2550. Found, 505.2559.

Preparation 30(1S,2S,4S,5R,6R)-2-[2′-tert-Butoxycarbonylamino-4′-(trityl-carbamoyl)-butyrylamino4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid diethyl ester

Prepare according to General Procedure A usingBoc-L-glutamine(Trityl)-OH (1.40 g, 2.86 mmoles) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (490 mg, 1.90 mmoles, Preparation 22), with theexception that DMAP is not used. Concentrate the crude reaction mixture,dissolve in dichloromethane, flash through a short silica gel plug withethyl acetate, and concentrate in-vacuo to give a yellow oil. Furtherpurify by silica gel chromatography eluting with 30% ethylacetate/hexanes to 100% ethyl acetate/hexanes to afford 1.3 g (94%) ofthe title compound.

[α]_(D) ²³=−21.28 (c=0.94, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.18-1.26 (6H, m), 1.44 (9H, s), 1.62-1.74(3H, m), 1.91-2.00 (1H, m), 2.05 (1H, m), 2.38-2.48 (3H, m), 2.54 (1H,m), 4.00-4.27 (6H, m), 7.18-7.30 (15H, m).

MS found 728.2 [M+H]⁺.

HRMS calcd for C₄₁H₄₉N₃O₉, 728.3547. Found, 728.3533.

Preparation 31(1S,2S,4S,5R,6R)-2-(2′S,6′-Bis-tert-butoxycarbonylamino-hexanoylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-lysine(Boc)-OH (910mg, 2.63 mmoles) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (450 mg, 1.75 mmoles, Preparation 22), with theexception that DMAP is not added to the reaction mixture. Concentratethe crude reaction mixture, dissolve in dichloromethane, flash through ashort silica gel plug with ethyl acetate, and concentrate in-vacuo togive a yellow oil. Further purify the material by silica gelchromatography eluting with ethyl acetate/hexanes (1:1) to afford 800 mg(78%) of the title compound.

[α]_(D) ²³=−30.19 (c=0.53, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.21-1.24 (6H, m), 1.26-1.77 (27H, m), 2.07(1H, m), 2.42 (1H, d, J=15.8 Hz), 2.59 (1H, dd, J=2.6, 5.5 Hz), 3.03(1H, t, J=6.2 Hz), 3.99 (1H, m), 4.07-4.28 (5H, m).

MS found 586.1 [M+H]⁺.

HRMS calcd for C₂₈H₄₇N₃O₁₀, 586.3340. Found, 586.3348.

Preparation 32(1S,2S,4S,5R,6R)-2-[2′-tert-Butoxycarbonylamino-3′-(trityl-carbamoyl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A usingBoc-L-Asparagine(Trityl)-OH (1.35 g, 2.84 mmoles) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (476 mg, 1.85 mmoles, Preparation 22), with theexception that DMAP is not added to the reaction mixture. Concentratethe crude reaction mixture, dissolve in dichloromethane, flash through ashort silica gel plug with ethyl acetate, and concentrate in-vacuo togive a yellow oil. Further purify the material by silica gelchromatography eluting with 20% ethyl acetate/hexanes to 50% ethylacetate/hexanes to afford 1.07 g (81%) of the title compound.

[α]_(D) ²³=−25.45 (c=0.55, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.19-1.26 (6H, m), 1.45 (9H, s), 1.61-1.69(2H, m), 2.05 (1H, m), 2.44 (1H, d, J=15.4 Hz), 2.51-2.72 (3H, m),4.02-4.21 (4H, m), 4.25 (1H, d, J=5.5 Hz), 4.36 (1H, dd, J=4.8, 8.8 Hz),7.18-7.30 (1SH, m).

MS found 714.1 [M+H]⁺.

HRMS calcd for C₄₀H₄₇N₃O₉, 714.3391. Found, 714.3380.

Preparation 33(1S,2S,4S,5R,6R)-2-[2′-tert-Butoxycarbonylamino-3′-(1′-tert-butoxycarbonyl-1H-indol-3′-yl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-tryptophan(Boc)-OH(1.18 g, 2.91 mmoles) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (520 mg, 1.90 mmoles, Preparation 22). Concentratethe crude reaction mixture, dissolve in dichloromethane, flash through ashort silica gel plug with ethyl acetate, and concentrate in-vacuo togive a yellow oil. Further purify the material by silica gelchromatography eluting with ethyl acetate/DCM (1:1) to afford 1.2 g(92%) of the title compound.

[α]_(D) ²³=−12.5 (c=0.96, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.18-1.27 (6H, m), 1.34 (9H, s), 1.62 (1H, t,J=2.9 Hz), 1.67 (9H, s), 1.74 (1H, dd, J=5.9, 15.2 Hz), 2.07 (1H, m),2.46 (1H, d, J=15.7 Hz), 2.60 (1H, d, J=2.9, 5.9 Hz), 2.89 (1H, dd,J=9.3, 15.0 Hz), 3.16 (1H, dd, J=4.9, 15.2 Hz), 4.04-4.12 (2H, m),4.18-4.24 (2H, m), 4.27 (1H, d, J=5.4 Hz), 4.43 (1H, dd, J=5.4, 9.3 Hz),7.26 (2H, m), 7.52 (1H, s), 7.64 (1H, d, J=7.3 Hz), 8.10 (1H, d, J=8.3Hz).

MS found 644.8 [M+H)⁺, 666.8 [M+Na)⁺.

HRMS calcd for C₃₃H₄₄N₃O₁₀, 666.3002. Found, 666.2988.

Preparation 34(1S,2S,4S,5R,6R)-2-(1′-tert-Butoxycarbonyl-pyrrolidine-2′S-carbonyl)₄-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-L-Proline and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 22). Purify the crude material on 110 gof silica on an ISCO system eluting with a gradient of 80% ethylacetate/hexanes to 100% ethyl acetate to afford 699 mg (84%) of thetitle compound.

[α]_(D) ²³=−52.53 (c=0.99, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.26 (6H, m), 1.44 (9H, s), 1.62 (1H, m),1.73(1H, dd, J=5.4, 15.3 Hz), 1.84 (1H, m), 1.97 (1H, m), 2.07 (4H, m),2.41 (1H, d, J=15.3 Hz), 2.50 (1H, m), 2.59 (1H, m), 4.12 (2H, m), 4.20(1H, q, J=7.4 Hz), 4.27 (1H, d, J=5.4 Hz).

MS found 454.9 [M+H]⁺, 476.8 [M+Na]⁺.

HRMS calcd C₂₂H₃₄N₂O₈Na, 477.2213. Found, 477.2210

Preparation 35(1S,2S,4S,5R,6R)-2-[2′S-tert-Butoxycarbonylamino-3′-(4-tert-butoxycarbonyloxy-phenyl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare the title compound according to General Procedure A using2S-tert-butoxycarbonylamino-3-(4-tert-butoxycarbonyloxy-phenyl)-propionicacid and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 22). Purify the crude material on 110 gof silica on an Isco system eluting with a gradient of 60% ethylacetate/hexane to 90% ethyl acetate/hexanes to afford 1.09 g (91%) ofthe title compound.

[α]_(D) ²³=−12 (c=1.0, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.25 (6H, m), 1.37 (9H, s), 1.52 (9H, s), 1.68(1H, dd, J=155.4, 15.3 Hz), 2.06 (1H, m), 2.38 (1H, d, J=15.3 Hz), 2.64(1H, m), 2.79 (1H, dd, J=8.4, 13.9 Hz), 3.05 (1H, dd, J=5.4, 13.9 Hz),4.20 (6H, m), 7.04 (2H, d, J=8.4 Hz), 7.26 (2H, d, J=8.4 Hz).

MS found 621.8 [M+H]⁺, 643.8 [M+Na]⁺.

HRMS calcd for C₃₁H₄₄N₂O₁₁Na, 643.2843. Found, 643.2845.

Preparation 36(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-4′-methylsulfanyl-butyrylamino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

The title compound was prepared according to General Procedure A using(Boc-L-methionine) and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 22). The crude material was purified on110 g of silica on an Isco system eluting with a gradient of 80% ethylacetate/hexanes to 100% ethyl acetate to afford 1.0 g (92%) of the titlecompound.

[α]_(D) ²³=−3° (c=1.0, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.26 (6H, m), 1.43 (9H, s), 1.84 (6H, m), 2.06(1H, m), 2.2 (1H, m), 2.48 (1H, m), 2.61 (1H, m), 3.37 (1H, m), 2.49(1H, m), 4.2 (6H, m).

MS found 488.8 [M+H]⁺, 510.8 [M+Na]⁺.

HRMS calcd for C₂₂H₃₆N₂O₈SNa, 511.2090. Found, 511.2071.

Preparation 37(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using commercially availableN-BOC-(L)-Alanine and(1S,2S,4S,5R,6R)-2-amino-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 22). Reflux reaction mixture overnight.Purify by prep HPLC, 1×500 g SiO₂ column, (10% ethyl acetate/hexanes to100% ethyl acetate). Yield 3.0 g (90%, 7.00 mmol) of a white foam.

[α]_(D) ²³=−32.31° (c=0.37, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.28 (6H, t, J=7.3 Hz), 1.34 (3H, d, J=7.3Hz), 1.46 (9H, s), 1.51-1.58 (2H, m), 1.64 (1H, s), 2.19 (1H, dd, J=3.3,6.2 Hz), 2.40 (1H, dd, J=2.9, 6.2 Hz), 2.80 (1H, d, J=15.8 Hz),4.10-4.36 (6H, m), 4.92 (1H, bs), 6.94 (1H, bs).

Anal Calcd for C₂₀H₃₂N₂O₈.0.1H₂O: C, 55.83; H, 7.54; N, 6.51. Found: C,55.57; H, 7.64; N, 6.44.

MS (ES) m/z 429.2 [M+H]⁺, 329.1 [M−Boc]+.

Preparation 38(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride

Purge a solution of(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (2.95 g, 6.88 mmol, Preparation 37) in ethyl acetate(30 mL) at 0° C. with anhydrous HCl gas until the solution is saturatedwith HCl. Stir the resulting reaction mixture at 0° C. until thereaction is judged complete by TLC. Purge the reaction mixture for 30minutes with N₂ to remove excess HCl gas. Concentrate the resultingsuspension to dryness in vacuo to afford 2.47 g (98%, 6.78 mmol) of thedesired amino hydrochloride salt. No further purification is necessary.

[α]_(D) ²³=−28.0° (c=0.50, MeOH).

¹H NMR (300 MHz, CDCl) δ 1.24 (3H, t, J=7.0 Hz), 1.28 (3H, t, J=7.3 Hz),1.50 (3H, d, J=7.3 Hz), 1.61 (1H, t, J=2.9 Hz), 1.76 (1H, dd, J=5.9,15.8 Hz), 2.10 (1H, dd, J=3.3, 5.9 Hz), 2.43 (1H, d, J=15.4 Hz), 2.60(1H, dd, J=2.9, 6.2 Hz), 3.90 (1H, q, J=7.0, 13.9 Hz), 4.15 (2H, q,J=7.3 Hz), 4.14-4.31 (3H, m).

Anal Calcd for C₁₅H₂₄N₂O₆.HCl.0.7H₂O: C, 47.73; H, 7.05; N, 7.42. Found:C, 47.96; H, 6.91; N, 7.04.

MS (ES) m/z 329.1 [M+H]⁺.

Preparation 39(1S,2S,4S,5R,6R)-2-[2′S-(2S-tert-Butoxycarbonylamino-propionylamino)propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddiethyl ester

Prepare according to General Procedure A using(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (0.2 g, 0.55 mmol, Preparation 38) andBoc-(L)-alanine (0.16 g, 0.82 mmol) with the exception that no DMAP isused. Purify using PC-TLC (ethyl acetate/hexanes) to yield 0.13 g(47.3%) of the title compound.

[α]_(D) ²³=−46.2 (c=0.52, CHCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.27 (3H, t, J=7.0 Hz), 1.28 (3H, t, J=7.0Hz), 1.36 (3H, d, J=7.0 Hz), 1.37 (3H, d, J=7.0 Hz), 1.44 (9H, s),1.58-1.65 (2H, m), 2.19 (1H, dd, J=3.0, 5.9 Hz), 2.46 (1H, dd, J=2.6,5.9 Hz), 2.70 (1H, d, J=15.4 Hz), 4.09-4.33 (7H, m), 4.48 (1H, app p,J=7.0 Hz), 5.05 (1H, bd, J=6.6 Hz), 6.79 (1H, bd, J=7.7 Hz), 7.26 (1H,s).

HRMS calcd for C₂₃H₃₈N₃O₉, 500.2608. Found, 500.2598.

Preparation 40(1S,2S,4S,5R,6R)-2-[2′S-(2-tert-Butoxycarbonylamino-acetylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure A using Boc-glycine (0.29 g, 1.6mmol) and(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (0.4 g, 1.1 mmol, Preparation 38) withthe exception that DMAP is not used. Purify using PC-TLC (ethylacetate/hexanes) to yield 0.14 g (26.2%) of the title compound.

[α]_(D) ²³=−14 (c=1.00, CDCl₃).

¹H NMR (300 MHz, CDCl₃) δ 1.27 (3H, t, J=7.3 Hz), 1.29 (3H, t, J=7.3Hz), 1.38 (3H, d, J=7.0 Hz), 1.45 (9H, s), 1.57-1.65 (2H, m), 2.19 (1H,dd, J=3.3, 5.9 Hz), 2.44 (1H, dd, J=2.9, 5.9 Hz), 2.74 (1H, d, J=15.8Hz) 3.70-3.86 (2H, m), 4.08-4.34 (6H, m), 4.56 (1H, app p, J=7.0 Hz),5.31 (1H, bs), 6.88 (1H, bd, J=7.0 Hz), 7.50 (1H, s).

HRMS calcd for C₂₂H₃₆N₃O₉, 486.2452. Found, 486.2444.

Preparation 41(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-Butoxycarbonylamino-4-methyl-pentanoylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure B using commercially availableN-BOC-(L)-leucine monohydrate and(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (0.2 g, 0.55 mmol, Preparation 38).Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanes to 100%ethyl acetate). Yield 0.54 g (62%, 1.00 mmol) of a white foam.

[α]_(D) ²³=−61.2° (c=0.49, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 0.93 (3H, d, J=6.2 Hz), 0.94 (3H, d, J=6.2Hz), 1.25 (3H, t, J=7.3 Hz), 1.27 (3H, t, J=7.3 Hz), 1.36 (3H, d, J=7.0Hz), 1.43 (9H, s), 1.53 (1H, d, J=9.5 Hz), 1.55-1.69 (5H, m), 2.18 (1H,dd, J=2.9, 5.9 Hz), 2.44 (1H, dd, J=2.9, 6.2 Hz), 2.70 (1H, d, J=15.8Hz), 4.06 (1H, bs), 4.13 (4H, q, J=7.3 Hz), 4.18-4.28 (2H, m), 4.31 (1H,d, J=5.9 Hz), 4.41-4.45 (1H, m), 4.85 (1H, bs), 6.57 (1H, d, J=7.3 Hz),6.97 (1H, bs).

Anal Calcd for C₂₆H₄₃N₃O₉.0.1H₂O: C, 57.46; H, 8.01; N, 7.73. Found: C,57.18; H, 8.00; N, 7.64.

HRMS calcd for C₂₆H₄₃N₃O₉ [M+Na]⁺, 564.2897. Found, 564.2922.

Preparation 42(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-Butoxycarbonylamino-3-methyl-butyrylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Prepare according to General Procedure B using commercially availableN-BOC-(L)-Valine and(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (0.2 g, 0.55 mmol, Preparation 38).Purify by PC-TLC, 4 mm SiO₂ rotor, (10% ethyl acetate/hexanes to 67%ethyl acetate). Yield 0.36 g (43%, 0.68 mmol) of a white foam.

[α]_(D) ²³=−65.5° (c=0.58, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 0.91 (3H, d, J=7.0 Hz), 0.97 (3H, d, J=6.6Hz), 1.27 (3H, t, J=7.3 Hz), 1.28 (3H, t, J=7.0 Hz), 1.37 (3H, d, J=17.3Hz), 1.44 (9H, s), 1.54-1.61 (3H, m), 2.12-2.20 (2H, m), 2.42 (1H, dd,J=2.6, 5.9 Hz), 2.70 (1H, d, J=15.4 Hz), 3.90 (1H, t, J=6.6 Hz), 4.13(2H, q, J=7.0 Hz), 4.20-4.29 (2H, m), 4.31 (1H, d, J=5.9 Hz), 4.40-4.48(1H, m), 4.93 (1H, bs), 6.41 (1H, d, J=7.3 Hz), 6.91 (1H, bs).

Anal Calcd for C₂₅H₄₁N₃O₉.0.2CH₂Cl₂: C, 55.58; H, 7.66; N, 7.72. Found:C, 55.76; H, 7.27; N, 7.49.

MS (ES) m/z 528.3 [M+H]⁺.

Preparation 43(1S,2S,4S,5R,6R)-2-(2′S-Acetylamino-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester

Stir a solution of(1S,2S,4S,5R,6R)-2-(2′S-amino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester hydrochloride (0.30 g, 0.82 mmol, Preparation 38) inCH₂Cl₂ (30 mL) at 0° C. as triethylamine (0.16 g, 1.6 mmol) then acetylchloride (0.10 g, 1.18 mmol) is added sequentially. Allow the reactionmixture to warm as it stirs overnight. Dilute the reaction mixture withethyl acetate (700 mL) and wash with aqueous NaHSO₄ and brine. Dry theorganic layer over magnesium sulfate. Purify by PC-TLC, 4 mm SiO₂ rotor,(10% ethyl acetate/hexanes to 100% ethyl acetate) to afford 0.24 g (79%,0.65 mmol) of a white foam.

[α]_(D) ²³=−48° (c=0.5, MeOH).

¹H NMR (300 MHz, CDCl₃) δ 1.27 (3H, t, J=7.3 Hz), 1.28 (3H, t, J=7.3Hz), 1.37 (3H, d, J=7.0 Hz), 1.55-1.62 (2H, m), 1.99 (3H, s), 2.19 (1H,dd, J=3.3, 6.6 Hz), 2.40 (1H, dd, J=2.6, 5.9 Hz), 2.77 (1H, d, J=15.8Hz), 4.07 (2H, q, J=7.0 Hz), 4.21-4.31 (2H, m), 4.33 (1H, d, J=6.2 Hz),4.43-4.47 (1H, m), 6.13 (1H, bs), 7.06 (1H, bs).

Anal Calcd for C₁₇H₂₆N₂O₇: C, 55.13; H, 7.08; N, 7.56. Found: C, 55.05;H, 7.12; N, 7.29.

HRMS calcd for C₁₇H₂₆N₂O₇ [M+Na]⁺, 393.1638. Found, 393.1644.

Preparation 44 3-acetoxy-2S-(tert-butoxycarbonylamino)propionic acid

Add N-methyl morpholine (2.8 g, 27.2 mmoles) and di-tert-butyldicarbonate (5.8 g, 25.2 mmoles) to a solution of O-acetyl-L-serine (3.7g, 25.2 mmoles) in 1:1 dioxane:water. Stir the reaction for 24 hoursthen partition between ethyl acetate and water. Extract the aqueous withethyl acetate and discard the organics. Adjust the pH to 0-1 withaqueous NaHSO₄. Extract the aqueous with ethyl acetate, dry over Na₂SO₄,filter, and concentrate in vacuo. Purify via flash chromatographyyielding 2.6 g (41.7%). Material was used in Preparation 15 withoutcharacterization.

Preparation 45(1S,5R)-3-(tert-Butoxycarbonylamino)-3-(tert-butoxycarbonyl)-6-oxabicyclo[3.1.0ρhexane

Add a solution of t-butyl methylmalonate (129 g, 0.75 mol) in THF (385mL) to a slurry of LiH (14.9 g, 1.875 mol) in THF (900 mL) andN,N-dimethylpropylene urea (DMPU, 155 g, 1.2 mol) over 30 minutes whilemaintaining the temperature at 0-5° C. Heat the reaction mixture to 65°C. and add a solution of cis-1,4-dichloro-2-butene (95%, 100 g, 0.8 mol,1.08 eq) in THF (100 mL) over 5.5 hours, maintaining the temperature at63-67° C. Stir the reaction for 4 hours at 65° C. A water/MTBE work-upof the reaction mixture yields1-(methoxycarbonyl)-1-(tert-butoxycarbonyl)cyclopent-3-ene.

¹H NMR (300 MHz, CDCl₃) δ 5.57 (s, 2H, CH═CH), 3.71 (s, 3H, CH₃), 2.95(s, 4H, 2CH₂), 1.42 (s, 9H, C(CH₃)₃).

IR (film) 1734 (C═O), 1646, 1268, 1149 cm⁻¹.

Cool the reaction solution containing1-(methoxycarbonyl)-1-(tert-butoxycarbonyl)cyclopent-3-ene to 10-15° C.and add to a cooled solution (10-15° C.) of 1 N NaOH (1.3 L, 1.3 mol)over 30 minutes. Stir the reaction solution at 25° C. for 24 hours andmonitored by GC assay. When the hydrolysis reaction is complete, addMTBE (645 mL) to the reaction mixture and stir the solution for 5minutes. Allow the layers to settle and separate. Discard the organiclayer. Add 1.5 M NaHSO₄ solution (1470 mL) to make the aqueous layeracidic (pH 2-3). Add MTBE (1.3 L) and separate the layers. Extract theaqueous layer with MTBE (385 mL) and wash the combined organic layerswith 5% LiCl solution. Concentrate the organic layer under vacuum anddilute with heptane (780 mL). Concentrate the solution to approximately500 mL and stir the resulting slurry 1 hour at ambient temperature.Filter the solid, wash with heptane (250 mL), and vacuum dry at 35° C.to give 103.29 g (61% yield) of 1-(carboxylicacid)-1-(tert-butoxycarbonyl)cyclopent-3-ene as a white solid.

mp=119° C.

¹H NMR (300 MHz, CDCl₃), δ 5.61 (s, 2H, CH═CH), 3.00 (s, 4H, 2CH₂), 1.46(s, 9H, C(CH₃)₃)—

IR: 3800-3000 (br, COOH), 1741 (CO₂R), 1705 (CO₂H), 1283), 1149 cm⁻¹.

Dissolve 1-(carboxylic acid)-1-(tert-butoxycarbonyl)cyclopent-3-ene (50g, 236 mmol) in 850 mL of 70:30 toluene:MTBE under nitrogen in a 1 Lflask with mechanical stirrer. Add thionyl chloride (33.6 g, 283 mmol,1.2 equiv) to the stirred reaction mixture, maintaining the temperatureat 23° C. Cool the reaction solution to 0-5° C. and add triethylamine(32.2 g, 318 mmol, 1.35 equiv) dropwise over 30 minutes. Warm thereaction mixture to 23° C. and stir for 1 hour. Add the reaction mixturerapidly dropwise to deionized water (625 mL), maintaining thetemperature at 20-25° C. Separate the layers and wash the organic layerwith 500 mL 1 M NaHCO₃ solution. Concentration of the organic layerisolates 1-(chlorocarbonyl)-1-(tert-butoxycarbonyl)cyclopent-3-ene as alight yellow liquid.

¹H NMR (300 MHz, CDCl₃) δ 5.61 (s, 2H, CH═CH), 3.04 (app q, J=15.1 Hz,4H, 2CH₂), 1.49 (s, 9H, C(CH₃)₃).

IR (film) 1796 (COCl), 1743 (CO₂R), 1274, 1233, 1158 cm⁻¹.

Add a solution of tetrabutyl ammonium hydrogen sulfate (0.81 g, 2.4mmol) in deionized water (700 mL) to sodium azide (20.16 g, 310 mmol).Add the solution containing1-(chlorocarbonyl)-1-(tert-butoxycarbonyl)cyclopent-3-ene inMTBE/toluene to the azide solution over 45 minutes. Stir the reactionmixture for 3 hours at 23° C. until the1-(chlorocarbonyl)-1-(tert-butoxycarbonyl)cyclopent-3-ene is consumed,as confirmed by GC analysis of the reaction solution. Separate thelayers and wash the organic layer with 1 M NaHCO₃ (540 mL) and deionizedwater (540 mL, then 270 mL). Dry the organic layer with Na₂SO₄ andfilter. Concentrate the solution under vacuum to yield 1-(acylazide)-1-(tert-butoxycarbonyl)cyclopent-3-ene as as an oil.

¹H NMR (300 MHz, CDCl₃) δ 5.58 (s, 2H, CH═CH), 2.96 (app t, J=2.3 Hz,4H, 2CH₂), 1.46 (s, 9H, C(CH₃)₃)—

IR (film) 2137 (CON₃), 1720 (CO₂R), 1246 (s, 1185, 1136 cm⁻¹.

Add the solution of 1-(acylazide)-1-(tert-butoxycarbonyl)cyclopent-3-ene over 1 hour to 110 mL oftoluene at 95° C. Evolution of nitrogen gas is addition rate controlledunder these conditions. Distill MTBE from the reaction during theaddition. Stir the reaction for 1 hour at 95° C., and allow to cool to23° C. overnight. Concentration of the solvent under vacuum yields1-(isocyanate)-1-(tert-butoxycarbonyl)cyclopent-3-ene as an oil.

¹H NMR (300 MHz, CDCl₃) δ 5.67 (s, 2H, CH═CH), 3.01 (d, J=15.6 Hz, 2H),2.61 (d, J=15.6 Hz, 2H), 1.50 (s, 9H, C(CH₃)₃).

IR (film) 2258 (—NCO), 1732 (—CO₂R), 1157 cm⁻¹.

Add t-butanol (35 g, 471 mmol) to a solution of potassium t-butoxide (1Min THF, 471 mL, 471 mmol) under nitrogen. Cool the reaction solution wascooled to 0-5° C. and add the toluene solution containing1-(isocyanate)-1-(tert-butoxycarbonyl)cyclopent-3-ene over 60 minutes,maintaining the temperature at 0-10° C. Warm the reaction to 23° C.,stir for 2 hours, and assay by GC for the disappearance of theisocyanate starting material. Add the reaction mixture to a mixture ofdeionized water (1.2 L) and MTBE (1.2 L) at 15° C. Stir the solution for20 minutes and separate the layers. Wash the organic layer with a 20%brine solution (250 mL) and separate the layers. Transfer the organiclayer concentrated via distillation, to approximately 250 mL. Addheptane (500 mL) and concentrate the solution to a total volume of 250mL. Cool the resulting slurry solution to 0° C., stir for 2 hours, andfilter. Wash the filter cake with cold heptane (2×100 mL) and vacuum dryto give 34.54 g (52% yield from 1-(carboxylicacid)-]-(tert-butoxycarbonyl)cyclopent-3-ene) of1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)cyclopent-3-ene as awhite solid.

m.p. 87-89° C.

¹H NMR (500 MHz, CDCl₃) δ 5.63 (s, 2H, CH═CH), 5.1 (bs, 1H, NH), 2.99(d, J=17.2 Hz, 2H), 2.57 (d, J=16.0 Hz, 2H), 1.46 (s, 9H), 1.44 (s, 9H).

¹³C NMR (CDCl₃) δ 173.3, 154.9, 127.7, 81.1, 64.5, 44.8, 28.3, 27.8.

IR (KBr): 3451, 2981, 2932, 1712, 1489, 1369, 1154 cm⁻¹.

MS (FIA) m/e (% relative intensity) 284.2 (M⁺+1, 56), 228.2 (73), 172.1(97), 128.0 (100).

Add a solution of1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)cyclopent-3-ene(20.00 g, 71 mmol) in 100 mL of EtOAc to a mixture of tetrabutylammoniumhydrogensulfate (4.08 g, 10 mmol, 0.17 equiv), magnesiummonoperoxyphthalate hydrate (MMPA, 52.4 g, 4.76% active oxygen=2.1equiv), and deionized water (100 mL). Stir the reaction was stirred witha mechanical stirrer for 19 hours. Add a solution of sodium sulfite (18g) in deionized water (100 L) to the reaction mixture over 30 minutes.Dilute the mixture with 100 mL of EtOAc and separate the layers. Washthe organic layer with deionized H₂O (100 mL), 2N NaOH (2×100 mL), anddeionized H₂O (2×100 mL). Distill the organic layer at 75-90° C. atatmospheric pressure until the volume reaches 40 mL. Cool the solutionto 75° C. and add hot heptane (120 mL). Cool the solution to 65° C. andoptionally seed with the desired product. Allow the resulting slurry tocool to ambient temperature and then cool in an ice bath for 1 hour.Filter the product and wash with 80 mL of cold 4:1 heptane:EtOAc, thenvacuum dry at 40° C. to obtain 15.45 g (73% yield) of the title compoundas a white solid. The compound may be further purified byrecrystallization from 2:1 isopropanol:water.

m.p. 130-133° C.

¹H NMR (500 MHz, CDCl₃) δ 5.0 (bs, 1H), 3.58 (s, 2H), 2.41 (d, J=15.3Hz, 2H), 2.20 (d, J=15.2 Hz, 2H), 1.43 (s, 9H), 1.40 (s, 9H).

¹³C NMR (CDCl₃) δ 171.3, 154.3, 81.2, 79.5, 62.8, 57.0, 38.6, 28.3,27.7.

IR (KBr): 3453, 2982, 2932, 1726, 1708, 1489, 1369, 1293, 1156, 840cm⁻¹.

MS (FIA) m/e (% relative intensity) 300.3 (M⁺+1, 65), 244.4 (68), 188.2(100) 144.1 (99).

Preparation 46 (1R,3R)-1,3-Di(methylbenzylamino)propane dihydrochloride

Heat (R)-α-methylbenzylamine (98% ee, 121 g, 1 mol) to 100° C. undernitrogen. Add dibromopropane (25.4 mL, 50.5 g, 250 mmol) dropwise over70 min. Heat the mixture for another 3 hours and then cool to 80° C. Addconcentrated (50%) NaOH solution (30 mL) dropwise over 10 minutes. Addwater (30 mL) to dissolve the solids and allow the mixture to cool toroom temperature over 30 minutes. Add MTBE (100 mL). Dissolve theprecipitate by adding 100 mL of water, and separate the layers. Wash theorganic layer with 50 mL of brine, dry (Na₂SO₄), and concentrate undervacuum to give 126.0 g of light yellow oil. Distill the oil under vacuumat 70° C. (head temperature), using a 12-inch column, to remove excess(R)-α-methylbenzylamine. Dissolve the pot residue (68 g of crudediamine) in 1 L of i-PrOH in a 2 L flask with a mechanical stirrer. Addconcentrated HCl (12 M, 45 mL, 540 mmol) dropwise over 10 minutes. Addan additional 100 mL of i-PrOH to ensure thorough mixing of the thickslurry. Stir the mixture for 90 minutes and filter. Wash the cake withi-PrOH and vacuum dry at 40° C. to obtain 64.32 g (72% yield, based ondibromopropane) of the title compound as a white solid.

¹H NMR (500 MHz, DMSO-d₆) δ 9.93 (bs, 2H), 9.47 (bs, 2H), 7.56 (d, J=7.0Hz, 4H), 7.37 (m, 6H), 4.27 (m, 2H), 2.84 (m, 2H) 2.48 (m, 2H), 2.1 (m,2H), 1.56 (d, J=6.7 Hz, 6H).

Preparation 47(1S)-1-(tert-Butoxycarbonylamino)-1-(tert-butoxycarbonyl)-cyclopent-4-en-3-one

Dissolve (1R,3R)-1,3-di(methylbenzylamino)propane dihydrochloride (24.65g, 69.3 mmol, Preparation 46) in 150 mL of water with stirring. Add tothe solution 35 mL (175 mmol) of 5 N NaOH solution, followed by 150 mLof MTBE. After stirring 15 minutes, separate the layers and extract theaqueous layer with 100 mL of MTBE. Wash the combined organic layers with100 mL of brine, dry with Na₂SO₄, and concentrate under vacuum to give18.06 g (92% yield) of 1R,3R-di(methylbenzylamino)propane as a colorlessoil.

In a 500 mL four-neck flask with overhead stirrer, dissolve 20.59 g(73.5 mmol) of 1R,3R-di(methylbenzylamino)propane in 30 mL each of dryMTBE and THF, under N₂ flow. Cool the solution to −45° C. and add 59 mL(147 mmol) of n-BuLi solution (2.5 M in hexanes) dropwise over 17minutes. Stir the light yellow solution for 2 hours at −45° C. Add asolution of(1S,3S,5R)-3-(tert-butoxycarbonylamino)-3-(tert-butoxycarbonyl)-6-oxabicyclo[3.1.0]hexane(10.00 g, 33.4 mmol) in 55 mL of dry THF dropwise over 28 minutes,keeping the temperature below −40° C. Rinse the addition funnel with anadditional 5 mL of THF. Stir the reaction mixture for 18 hours at −45°C. and then quench via the dropwise addition of 4 N aqueous sulfuricacid (75 mL). Remove the cooling bath after the acid addition iscomplete. Separate the layers and extract the aqueous layer with 50 mLof MTBE. Wash the combined organic layers with 50 mL each of water andbrine, dry with Na₂SO₄, and concentrate under vacuum to give 9.78 g ofcrude 578242 as a white solid. Dissolve the solid in 33 mL of hot MTBEand add heptane (66 mL) portionwise. Allow the stirred solution to coolto room temperature, then stir the mixture for 1 hour at 0° C. Filterthe solid, wash with 2×10 mL of cold 2:1 heptane:MTBE, and vacuum dryfor 4 hours at 35° C. to provide 8.52 g (85% yield) of(1S,3R)-1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)-3-hydroxycyclopent-4-eneas a white solid.

m.p. 111-112 C.

Chiral HPLC assay: 99.7% ee.

[α]_(D)=+114 (c 1, MeOH.

¹H NMR (500 MHz, CDCl₃) δ 6.1 (bs, 1H), 5.9 (bs, 1H), 5.55 (d, J=5.0 Hz,1H), 4.8 (m, 1H), 4.44 (d, J=10.5 Hz, 1H), 2.87 (dd, J=14.5, 7.5 Hz,1H), 2.00 (d, J=14.5 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 9H).

IR (KBr): 3413, 2983, 1703, 1491, 1370, 1309, 1255, 1155, 1055 cm⁻¹.

MS (FIA) m/e (% relative intensity) 300.3 (M⁺+1, 15), 226.2 (29), 170.1(100), 126.1 (89), 108.3 (20).

Add 2,2,6,6-tetramethyl-1-piperdinyloxy (free radical) (TEMPO) (0.84 g,5.3 mmol) and KBr (0.63 g, 5.3 mmol, in 2 mL of water) to a solution of(1S,3R)-1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)-3-hydroxycyclopent-4-ene(20 g, 66.8 mmol) in MTBE (200 mL). Cool the reaction mixture to 0° C.and add a solution of NaOCl (3.14%, 240 g, 100 mmol) containing NaHCO₃(8.4 g) dropwise, keeping the temperature below 5° C. Stir the reaction1 hour at 0° C. and allow to warm to room temperature. Separate thelayers and extract the aqueous layer with MTBE (2×200 mL). Wash thecombined organic layers with 200 mL of 1N HCl solution containing 2.21 gof KI followed by 10% Na₂SO₃ solution (200 mL). Wash the organic layerwith water (2×200 mL) and concentrate to dryness under vacuum. Dissolvethe crude product in MTBE (60 mL) at 50° C. and crystallize by additionof heptane (200 mL) over 1 h. Cool the mixture was cooled to 0° C. over2 hours and then filter. Wash the filter cake with 100 mL of coldheptane:MTBE (65:35) and vacuum dry to give 16.99 g (89% yield) of thetitle compound as a white solid.

m.p. 116-18° C.

[α]_(D)=+123 (c 1, MeOH).

¹H NMR (500 MHz, CDCl₃) δ 7.4 (bs, 1H), 6.32 (d, J=5.5 Hz, 1H), 5.6 (bs,1H), 2.87 (d, J=18.2 Hz, 1H), 2.9 (d, J=18.2 Hz, 1H), 1.43 (s, 18H). ¹³CNMR (CDCl₃) δ 206.1, 170.2, 160.8, 154.9, 136.1, 84.5, 66.1, 46.6, 28.9,28.4.

IR (KBr): 3419, 2983, 1722, 1487, 1730, 1300, 1259, 1151, 1012 cm⁻¹.

MS (FIA) m/e (% relative intensity) 254.2 (M⁺+1, 11), 242.3 (18), 228.2(13), 186.1 (76), 143.2 (11), 242.3 (100).

Preparation 48 1-(2-tert-butoxy-2-oxoethyl)tetrahydrothiophenium bromide

Add t-butyl bromoacetate (2.44 L, 16.52 mole, 1 eq) to a solution oftetrahydrothiophene (2.19 L, 24.8 mole, 1.5 eq) in acetone (11.38 L), ina 22 L flask over 30-60 minutes while maintaining the temperaturebetween 15-25° C. using a water bath. Stir the reaction for 22 hours andassay a sample by ¹H NMR to confirm the completion of the reaction.Filter the precipitate, wash with acetone (2 L), and vacuum dry for 3days at 28-33° C. to give 4.328 Kg (92.5% yield) of the title compound.

¹H NMR (500 MHz, DMSO d-6) δ 4.40 (s, 1H), 3.51 (m, 2H), 3.48 (m, 2H),2.23 (m, 2H), 2.13 (M, 2H), 1.42 (s, 9H).

Preparation 49(1S,2S,5R,6R)-2-(tert-Butoxycarbonylamino)-4-oxo-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester

Add KOtBu (42 mL, 42 mmol, 1 M solution in THF, 2.5 equiv) to a 0° C.solution of 1-(2-tert-butoxy-2-oxoethyl)tetrahydrothiophenium bromide(11.9 g, 42 mmol, 2.5 equiv., Preparation 48) in acetonitrile (30 mL)under N₂ over 10 minutes keeping the temperature below 5° C. Stir themilky solution cold for 1.5 hours. Add trifluoroethanol (6.9 g, 69 mmol,4.1 equiv) dropwise. Add a solution of(1S)-1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)-cyclopent-4-en-3-one(5 g, 16.8 mmol, Preparation 47) and trifluoroethanol (13.3 g, 132 mmol,7.9 equiv) in acetonitrile (20 mL), over 5 minutes, maintaining thetemperature at 3-5° C. Stir the solution for 4.5 hours at 0-5° C. AddMTBE (155 mL) and H₂O (80 mL) to the cold reaction mixture. Separate thelayers and wash the organic layer with H₂O (50 mL), then 20% brine (50mL). Concentrate the organic layer via atmospheric pressure distillationto approximately 30 mL. Add heptane (100 mL) and concentrate thesolution. Add additional heptane as needed until the vapor temperatureof the distillate reaches 93° C. Add THF (65 mL) to yield a solution ofthe title compound.

Preparation 50(1S,2S,5R,6R)-2-(tert-Butoxycarbonylamino)-4-oxo-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester

Add 1.7 L of saturated aqueous K₂CO₃ solution to a 0° C. solution of1-(2-tert-butoxy-2-oxoethyl)tetrahydrothiophenium bromide (757 g, 2.67mol, Preparation 48) in 2.2 L of CH₂Cl₂, keeping the temperature below10° C. After stirring the biphasic mixture 1.5 hours, add 223 mL of 50%NaOH solution portionwise, keeping the temperature below 5° C. Stir themixture 3 hours and filter. Rinse the salts with CH₂Cl₂. Separate thelayers and extract the aqueous layer with 600 mL of CH₂Cl₂. Dry thecombined organic layers over solid K₂CO₃ and concentrate under vacuum togive 533.3 g (98% yield) of(2-tert-butoxy-2-oxoethyl)tetrahydrothiophenium as a pale yellow oil.Upon storage in the freezer, the oil crystallizes giving an off-whitesolid.

mp 48-50° C.

¹H NMR (500 MHz, CDCl₃) δ 3.12 (m, 2H), 3.00 (m, 2H), 2.85 (s, 1H), 2.41(m, 2H), 1.84 (m, 2H), 1.38 (s, 9H).

Add 474 mL (6.5 mol, 10 equiv) of trifluoroethanol to a 0° C. solutionof(1S)-1-(tert-butoxycarbonylamino)-1-(tert-butoxycarbonyl)-cyclopent-4-en-3-one(194 g, 653 mmol) in 650 mL of CH₂Cl₂. Add a solution of(2-tert-butoxy-2-oxoethyl)tetrahydrothiophenium (396 g, 1.96 mol) in 325mL CH₂Cl₂ dropwise over 40 minutes keeping the temperature below 10° C.Remove the ice bath after 1 hour. Add deionized water (680 mL) andseparate the layers. Extract the aqueous layer with 400 mL CH₂Cl₂. Washthe combined organic layers with 500 mL of brine, dry with Na₂SO₄, andconcentrate under vacuum to obtain 587 g of an amber, oily solid.Dissolve the solid in 400 mL of CH₂Cl₂ and elute through a 1.6 Kg silicagel plug using 5:1:1 hexanes: methyl t-butylether:CH₂Cl₂ as eluent toyield a total of 13.2 L of eluent. Concentrate the eluent to give 398.7g of white solid. Dissolve the solid in 3 L of refluxing 70:30 hexanes:MTBE. Allow the solution to cool to room temperature overnight, thencool in an ice bath for 1 hour. Filter the solid, rinse with coldsolvent (approximately 700 mL), and vacuum dry at 35° C. to obtain 173 g(64% yield) of(1S,2S,5R,6R)-2-(tert-butoxycarbonylamino)-4-oxo-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester as a white solid.

mp 144-46° C.

[α]_(D)=+30.5 (c 1, CHCl₃).

¹H NMR (500 MHz, CDCl₃) δ 5.36 (d, 1H), 2.88 (m, 1H), 2.64 (dd, J=5.2,3.2 Hz, 1H), 2.37 (d, J=2.7 Hz, 1H), 2.23 (bs, 1H), 1.45 (s, 9H), 1.43(s, 18H).

¹³C NMR (125 MHz, CDCl₃) δ 206.2, 171.2, 168.5, 155.3, 83.4, 82.7, 80.7,61.2, 43.2, 36.0, 34.3 28.4, 28.2, 28.0, 25.3.

IR (CHCl₃): 2982, 1744, 1719, 1485, 1394, 1309 cm⁻¹.

MS (ES+) m/e (% relative intensity) 412.2 (M⁺+1, 79), 356.2 (50), 300.1(97), 2λ^(6.1) (68), 244.1 (100). Anal. Calcd. for C₂₁H₃₃NO₇ (411.29):C, 61.30; H, 8.08; N, 3.40. Found: C, 61.32; H, 8.04; N, 3.51.

Preparation 51(1S,2S,4S,5R,6R)-2-(tert-Butoxycarbonylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester

Cool the solution from Preparation 49 to 0° C. under nitrogen and addlithium tri-sec-butylborohydride (L-Selectride™, 1 M in THF, 21 mL, 21mmol) dropwise. Stir the reaction mixture for 45 minutes at 0° C. Add asolution of 2 M sodium carbonate (31 mL) dropwise, keeping thetemperature below 8° C. Add a solution of 30% H₂O₂ (7.15 g, 63 mmol) in20 mL of water, keeping the temperature below 150C. After stirring 10min, add MTBE (210 mL) and deionized water (100 mL). Separate the layerswere separated and wash the organic layer with saturated Na₂SO₃ solution(40 mL) and 1 M NaHSO₄ solution (40 mL). Concentrate the organic layerby distillation at atmospheric pressure to a volume of approximately 90mL. Continue the distillation and add heptane to maintain a constantvolume. When the vapor temperature of the distillate reaches 930C, coolthe solution to 70° C. and add 7 mL of THF. Cool the solution to 0° C.and stir for 1 hour. Filter the solid, wash with cold heptane (10 mL),and vacuum dry at 50° C. to obtain 4.68 g (67% yield) of(1S,2S,4S,5R,6R)-2-(tert-butoxycarbonylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester as a white solid.

m.p. 187-88° C.

¹H NMR (CDCl₃, 500 MHz) δ 5.31(bs, 1H), 4.38 (d, J_(AB)=10.5 Hz, 1H),4.30 (dd, J=11.0, 6.0 Hz, 1H), 2.68 (d, J=15.3 Hz, 1H), 2.17 (m, 1H),2.07 (m, 1H), 1.58 (m, J=15.2 Hz, 1H), 1.45 (s, 9H), 1.44 (s, 9H), 1.43(s, 9H).

¹³C NMR (75 MHz, CDCl₃) δ 175.2, 170.5, 155, 83.2, 81.3, 80.1, 73.7,66.9, 43.1, 36.0, 34.4, 29.3, 28.3, 28.1, 27.4, 22.1.

IR (CHCl₃): 3445, 2982, 1714, 1485, 1361 cm⁻¹.

MS (ES+) m/e (% relative intensity) 414.2 (M⁺+1, 58), 358.1 (75), 302.1(78), 246.0 (100).

[α]_(D)=−28.6 (c 1, MeOH).

Preparation 52(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-Butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Add a solution of(1S,2S,4S,5R,6R)-2-(tert-butoxycarbonylamino)₄-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester (25.00 g, 60.5 mmol, Preparation 51) in CH₂Cl₂(230 mL) to a −78° C. solution of Deoxofluor™ (16.05 g, 72.6 mmol, 1.2equiv) in CH₂Cl₂ (105 mL) over 2 h. Stir the reaction mixture for 1.5hours, and add an additional 1.66 g of Deoxofluor™. Stir the solution 30minutes and allow to warm to −10° C. Add a solution of saturated NaHCO₃(105 mL) dropwise over 20 minutes, keeping the temperature below 5° C.Adjust the pH of the aqueous layer to 7 by adding 160 mL of saturatedNaHCO₃ solution. Allow the mixture to warm to ambient temperature andseparate the layers. Extract the aqueous layer with 100 mL of CH₂Cl₂.Wash the combined organic layers with 250 mL of brine, then dry withNa₂SO₄. Remove the solvent under vacuum and add heptane (75 mL) to thecrude product. Heat the resulting mixture to 50° C. until all solids aredissolved and stir at ambient temperature for 24 hours. Cool the mixtureto 0° C. in an ice bath and filter. Rinse the product with cold heptaneand vacuum dry to give 20.23 g (80% yield) of(1R,2S,4R,5R,6R)-2-(tert-butoxycarbonylamino)-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester as a white solid.

m.p. 140-143° C.

[α]_(D)=+20.6 (c 1, CHCl₃).

¹H NMR (CDCl₃, 500 MHz) δ 5.45 (dd, J_(H-F)=56, J_(H-H)=4.8 Hz, 1H),5.28 (bs, 1H), 3.00 (m, 1H), 2.23 (bs, 1H), 2.11 (m, 1H), 2.08 (m, 1H),1.46 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H), 1.37 (m, 1H).

¹³C NMR (CDCl₃, 75 MHz) δ 177.7, 170.5, 155.0, 94.4, 92.0, 82.2, 81.5,80.2, 64.7, 37.7, 33.2, 29.7, 29.3, 28.3, 28.1, 27.8, 20.4.

MS (ES+) m/e (% relative intensity) 416.2 (M⁺+1, 66), 360.1 (67), 304.1(100), 248.0 (60).

IR (CHCl₃): 3444, 2981, 1715, 1485, 1369 cm⁻¹.

Add SOCl₂ (21.29 g, 13.05 mL, 0.179 mole, 5 eq) to a solution of(1R,2S,4R,5R,6R)-2-(tert-butoxycarbonylamino)-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid di-tert-butyl ester (14.87 g, 0.036 mole) in EtOH (abs, 149 mL)dropwise over 10 minutes without cooling to produce a gently refluxingsolution. Reflux the solution overnight. Remove the solvent from thereaction under vacuum. Dissolve the residue in EtOAc (150 mL) and add asolution of 10% Na₂CO₃ (75 mL) dropwise over 5-10 minutes with stirring.Separate the layers and extract the aqueous layer with EtOAc (50 mL).Wash the combined organic extracts with brine (1×50 mL), dry overNa₂SO₄, filter and concentrate the product,(1R,2S,4R,5R,6R)-2-amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester, in vacuo to a thick liquid which solidifies uponstanding (11.02 g).

Add N-methylmorpholine (22.44 mL, 204 mmol) to a solution ofN-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL of methylene chloride at−22° C. under nitrogen, followed by the addition of iso-butylchloroformate (26.48 mL, 204 mmol) dropwise over 15 minutes such thatthe reaction temperature does not exceed −18° C. Stir the resultant thinslurry at −20° C. for 30 minutes, then add a solution of(1R,2S,4R,5R,6R)-2-amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (49.46 g, 191 mmol) in 247 mL of methylene chlorideover 40 minutes such that the reaction temperature does not exceed −16°C. Remove the reaction from the cooling bath and stir at ambienttemperature for 70 minutes. Add 408 mL of 1 N hydrochloric acid, stirfor 5 minutes, and separate the layers. Wash the organic layer withsaturated aqueous sodium bicarbonate (1×408 mL), dry (Na₂SO₄), filter,and concentrate the product,(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester, in vacuo to a white foam (88.16 g).

Add 46.7 mL (93.4 mmol) of 2N sodium hydroxide to a solution of crude(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (17.5 g, 37.3 mmol theoretical) in 46.6 mL oftetrahydrofuran at room temperature. Stir the biphasic mixturevigorously at room temperature until homogeneous, then stir for anotherhour (three hours total). Dilute the mixture with 46 mL of t-butylmethyl ether, then mix for 10 minutes and separate the layers. In aseparate flask, add 93 mL of water, then 8.4 mL (101 mmol) ofconcentrated HCl. Optionally add(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid seed crystals to the acid solution, followed by the aqueous layerfrom above. Add the aqueous layer slowly at first, so that a moderatelythick slurry forms. At this time, increase the rate of addition (40minutes total addition time). Rinse the addition funnel with water (16mL). Stir the resultant slurry for 2 hours, filter, wash with water(2×32 mL), and vacuum dry at 45° C. to constant weight to provide 13.9 g(99%) of the title compound as a white solid.

Preparation 53(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester

Add thionyl chloride to a slurry of(1R,4S,5S,6S)-4-Amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (10 g, 42.5 mmol, U.S. Pat. No. 5,688,826) in 100 mL of 2B ethanolat room temperature (15.5 mL, 212.6 mmol) dropwise over 20 minutesfollowed by rinsing with 40 mL of ethanol. Heat the slurry to reflux andstir overnight. Cool the resultant solution to room temperature andconcentrate to a gelatinous residue. Add ethyl acetate (50 mL) to theresidue, followed by dilution with another 94 mL of ethyl acetate.Slowly add 15% aqueous sodium carbonate (70 mL) to the mixture withswirling by hand to gradually afford dissolution giving a final pH of7.95. Filter and separate the layers. Extract the aqueous layer withethyl acetate (2×100 mL). Wash the combined organic extracts with brine(1×100 mL), dried (MgSO₄), filter, and concentrate in vacuo to providethe title compound as a faint yellow oil that solidifies to an off-whitesolid (11.71 g, 95% yield).

m.p. 80-83° C.

[α]²⁵ _(D) −57.7 (c 1.04, CH₃OH).

500 MHz ¹H NMR (CDCl₃) & 4.31 (q, 2H, J=7.0 Hz), 4.20 (m, 2H), 3.78 (d,1H, 3=15.0 Hz), 3.36 (dd, 1H, J=4.0, 7.0 Hz), 2.93 (dd, 1H, J=4.0, 7.0Hz), 2.81 (d, 1H, J=15.0 Hz), 2.46 (t, 1H, J=4.0), 1.34 (t, 3H, J=7.0),1.30 (t, 3H, J=7.0).

¹³C NMR (125 MHz, CD₃Cl₃) δ 171.68, 168.57, 63.26, 62.42, 59.96, 56.06,43.78, 32.25, 22.49, 14.31, 14.25.

FTIR (ATR) 3364.15 (s), 1725.95 (s), 1304.91 (s), 1259.24 (s), 1200.84(s), 1104.91 (s), 1022.99 (s), 896.45 (s), 851.21 (s) cm⁻¹.

Anal. Cald for C₁₁H₁₇NO₆S: C, 45.35; H, 5.88; N, 4.81. Found: C, 45.02;H, 5.75; N, 4.82.

Preparation 54 (1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester

Add N-methyl morpholine (14.4 mL, 130.9 mmol) to a clear solution ofN-Boc-L-methionine (32.64 g, 130.9 mmol) in 110 mL of methylene chlorideat −22° C. under nitrogen, followed by addition of iso-butylchloroformate (17 mL, 130.9 mmol) dropwise over 7 minutes to maintainthe reaction temperature at −22° C. Upon completion of the addition, aresultant thin slurry was forms. Stir at −22 to −26° C. for 30 minutes.Add a solution of(1R,4S,5S,6S)-4-amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester (35.65 g, 122.4 mmol, Preparation 53) in 107 mL ofmethylene chloride during 15 minutes, followed by a rinse of 36 mL ofmethylene chloride. Remove the reaction from the cooling bath and stirat room temperature for 70 minutes Add 51 mL of 5 N hydrochloric acid tothe solution, then separate the layers. Back extract the aqueous layerwith methylene chloride (2×10⁷ mL). Wash the combined organic layerswith saturated aqueous sodium bicarbonate (1×10⁷ mL), dried (MgSO₄),filter, and concentrate in vacuo to furnish 65.82 g (103% weight yield)of the title compound as a white foam.

[α]²⁵ _(D) −12.7 (c 1.2, CH₃OH).

500 MHz ¹H NMR (CDCl₃) δ 7.53 (s, 1H), 5.06 (d, 1H, J=8.0 Hz), 4.34-4.20(m, 6H), 3.41 (dd, 1H, J=4.0, 7.0), 2.97-2.89 (m, 2H), 2.64-2.59 (m, 2H,J=4.0), 2.12-1.89 (m, 5H), 1.47 (s, 9H), 1.32 (t, 6H, J=7.0).

¹³C NMR (125 MHz, CD₃OD) δ 172.53, 169.03, 167.88, 156.00, 80.62, 63.45,62.56, 60.20, 55.33, 52.78, 42.81, 31.52, 31.38, 30.12, 28.49, 22.69,15.44, 14.23, 14.143.

FTIR (ATR) 3341.88 (w), 2979.38 (s), 1733.03 (s), 1674.92 (s), 1514.58(s), 1315.80 (s), 1255.15 (s), 1161.47 (s), 1142.63 (s), 1025.68 (s),854.85 (s), 763.53 (s) cm⁻¹.

Preparation 55(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid monosodium salt

Add 141 mL (282 mmol) of 2N sodium hydroxide to a solution of(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester (58.95 g, 112.8 mmol theoretical, Preparation 54) in141 mL of tetrahydrofuran at room temperature. Stir the mixturevigorously at room temperature for two minutes. Dilute the solution with141 mL of tert-butyl methyl ether, then separate the layers. Furtherdilute the aqueous layer with 141 mL of water and add concentratedhydrochloric acid dropwise to lower the pH to 4.46. Stir for 10 minutesto obtain a thin slurry. Add more concentrated hydrochloric acid to theslurry to drop the pH to 1.4 (total use of 17 mL of conc. HCl, 204mmol). After stirring for 2 hours, filter the slurry. Wash the cake withwater (2×118 mL) and dry in vacuo at 45° C. for 1 hour beforetransferring to a weighing pan. Dry the cake again in vacuo at 45° C.for 16 hours and at 58° C. for 5 hours to provide 52.96 g (96% weightyield) of the title compound as a white solid.

mp (decomposed) 258° C.

[α]²⁵ _(D) −25.2 (c 1.03, H₂O).

500 MHz ¹H NMR (D₂O) δ 4.07-4.01 (m, 2H), 3.45-3.43 (m, 1H), 3.11 (d,1H, J=15.0 Hz), 2.85 (m, 1H), 2.71 (s, 3H), 2.47-2.35 (m, 3H), 1.96-1.90(m, 4H), 1.78-1.72 (m, 1H), 1.28 (s, 9H).

¹³C NMR (125 MHz, CD₃OD) δ 174.45, 173.58, 172.80, 157.46, 81.71, 61.41,55.04, 53.24, 42.29, 31.71, 30.85, 29.41, 27.79, 23.65, 14.41.

FTIR (ATR) 3287.62 (s), 1698.00 (s), 1528.91 (s), 1327.36 (s), 1283.74(s), 1245.90 (s), 1174.81 (s), 1109.06 (s), 1053.05 (s), 874.27 (s),808.95 (s) cm⁻¹.

Preparation 56(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid

Add 397 mL (795 mmol) of 2N sodium hydroxide to a solution of(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid diethyl ester (166.15 g, 318 mmol, Preparation 54) in 480 mL oftetrahydrofuran at room temperature. Stir the mixture vigorously at roomtemperature for two minutes, at which time the reaction becomeshomogeneous to form a clear faint yellow/green solution. Stir for anadditiona two hours at room temperature. Dilute the solution with 480 mLof tert-butyl methyl ether, then separate the layers. Add the aqueouslayer dropwise to a solution of concentrated hydrochloric acid (71.5 mL,858 mmol) in water (960 mL). Add ethyl acetate (500 mL) followed by therest of the aqueous layer to result an emulsion, and further dilute withethyl acetate (460 mL). Stir the emulsion for 40 minutes, filter, andwash with water (2×250 mL). Separate the layers of the filtrate and backextract the aqueous layer with ethyl acetate (500 mL). Wash the combinedorganic layers with brine (75 mL), dry (MgSO₄), filter, and concentratein vacuo to provide 125.26 g (84% corrected yield) of the title compoundas a white foam.

Preparation 57 Ethyl 2-Bromo-2-fluoro-2-(3-oxocyclopentyl)acetate

Add 8.45 mL (50.4 mmol) of triethylsilyl chloride to a suspension of2.99 g of activated Zn (45.8 mmol) in anhydrous acetonitrile (100 mL) at−20° C. and stir the mixture for 5 minutes. Add 8.0 mL (57.3 mmol) ofethyl 2,2-dibromo-2-fluoracetate and stir the mixture for 90 minutes at−20° C. Add 1.86 mL (22.9 mmol) of 2-cyclopenten-1-one, and stir thereaction mixture overnight, allowing the temperature to rise slowly toroom temperature. Add HCl 1N (125 mL) and EtOAc (100 mL). Wash theorganic layer with saturated NaHCO₃ (2×150 mL), water (2×1150 mL) andbrine (2×1150 mL), dry over anhydrous MgSO₄, filter and concentrateunder reduced pressure. Purify the residue by column chromatographyusing EtOAc/hexane (1:8) as eluent to give the title compound (5.36 g,88% overall yield) as a colorless oil as a mixture of diastereoisomers.

¹H-NMR (300 MHz, CDCl₃): 1.34-1.41 (m, 6H), 1.91-1.95 (m, 1H), 2.04-2.68(m, 1H), 3.12-3.31 (m, 2H), 4.33-4.43 (m, 4H).

Zn activation: Add 10 mL of concentrated HCl to a suspension of 100 g ofZn dust in water (900 mL). Stir the mixture for 20 minutes at roomtemperature. Decant the water, and wash the residue with water (3×250mL), acetone (3×1 50 mL), and ether (2×100 mL). Dry the residue underreduced pressure at 35° C. overnight.

Preparation 58 Ethyl(1RS,5RS,6RS)-6-fluoro-2-oxobicyclo[3.1.0]hexane-6-carboxylate

Add 13.0 mL (75.0 mmol) of ethyldiisopropylamine to a solution of 2.0 g(7.5 mmol) of ethyl 2-bromo-2-fluoro-2-(3-oxocyclopentyl)acetate(Preparation 57) in DMF (8 mL), at 0° C., and stir the mixture overnightat room temperature. Add a solution of HCl 1N (20 mL), water (15 mL),and EtOAc (75 mL). Extract the organic layer, wash with saturated NaHCO₃(2×100 mL), water (2×100 mL), and brine (2×100 mL), dry over anhydrousMgSO₄, filter and concentrate under reduced pressure. Purify the residueby column chromatography using EtOAc/hexane (1:4) as eluent to give thetitle compound (1.17 g, 84% yield) as a mixture trans:cis 5:1 of isomersas a colorless oil.

¹H-NMR (300 MHz, CDCl₃): 1.33 (t, 3H, J=7.1 Hz), 2.19-2.34 (m, 3H),2.41-2.49 (m, 1H), 2.59 (d, 1H, J=6.6 Hz), 2.71-2.76 (m, 1H), 4.29 (q,2H, J=7.1 Hz).

Preparation 59(1RS,2SR,5RS,6RS)-2-Spiro-5′-hydantoin-6-fluoro-bicyclo[3.1.0]hexane-6-carboxylicacid

Stir a mixture of ethyl (1RS, 5RS,6RS)-6-fluoro-2-oxobicyclo[3.1.0]hexane-6-carboxylate (0.1 g, 0,54 mmol,Preparation 58) and 1N NaOH (0.55 mL, 0,55 mmol) in EtOH (1 mL) for 10min with ice cooling. Add 1 N HCl to the mixture dropwise until pH 1,and partition the resulting mixture between EtOAc and brine. Extract theaqueous phase with EtOAc twice, and dry the combined organic layers overMgSO₄ and then concentrated under reduced pressure. Stir a mixture ofthe residue, (NH₄)₂CO₃ (0.31 g, 3.2 mmol), and KCN (0.11 g, 1,62 mmol)in EtOH and water (1:1) (2 mL) at 60° C. overnight. Cool in an ice bathand acidify the mixture by treatment with 1 N KHSO₄. Remove the solventunder reduced pressure and redissolve the residue in MeOH, filter andconcentrate in vacuo. The crude may be used without furtherpurification.

Preparation 60(1R,2S,5R,6R)-Diethyl-2-(2′R-tert-butoxycarbonylamino-propionylamino)-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylateand(1S,2R,5S,6S)-Diethyl-2-(2′R-tert-butoxycarbonylamino-propionylamino)-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylate

Add 1.59 g (5.0 mmol) of Ba(OH)₂ to a solution of 0.32 g (1.5 mmol) of(1R*,2S*,5R*,6R*)-2-spiro-5′-hydantoin-6-fluoro-bicyclo[3.1.0]hexane-6-carboxylicacid (Preparation 59) in water (10 mL), and stir the mixture at 1050Covernight. Acidify the solution to pH 1 with 1N HCl at 0° C., thenconcentrate under reduced pressure. Redissolve the residue andconcentrate under reduced pressure with absolute EtOH several timesuntil the solid is perfectly dried. Add 0.37 mL (5.0 mmol) of SOCl₂ tothe residue dissolved in absolute EtOH (20 mL) at 0° C., then stir themixture at reflux for 5 hours. Basify the solution with saturated NaHCO₃and add EtOAc (25 mL). Separate the organic layer, and extract theaqueous layer with more EtOAc (2×25 mL). Dry the combined organic layersover anhydrous MgSO₄, filter and concentrate at reduced pressure.Dissolve the residue in 20 mL of a mixture of DCM-DMF (4:1), 0.73 g (1.9mmol) of HATU, 0.26 g (1.9 mmol) of HoAt, 0.35 g (1.8 mmol) of L-Ala.Add 2.7 mL (15.3 mmol) of diisopropylethylamine and stir the mixtureunder Ar overnight. Add DCM (15 mL), separate and wash with saturatedNaHCO₃ (2×25 mL), water (2×25 mL), and brine (2×25 mL). Wash the organiclayer over anhydrous MgSO₄, filter, and concentrate at reduced pressure.Purify the residue by column chromatography using EtOAc/hexane (1:2) aseluent to give the title compound (0.33 g, 51% overall yield) as amixture of 1:1 of diastereomers as colorless oil.

The mixture of diastereomers were separated by chiral HPLC using thefollowing analytical method: Chiralpak AD 10 m, 4.6×250 mm; Eluent: 10%IPA in Hexane; Flow: 1.0 mL/min; UV: 215 nm. Isomer A retention time=5.9min. Isomer B retention time=9.2 min.

Isomer A:(1R,2S,5R,6R,2′R)-Diethyl-2-(2′-tert-butoxycarbonylamino-propionylamino)-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylate

¹H-NMR (300 MHz, CDCl₃): 1.18 (t, 3H, J=7.1 Hz), 1.23 (t, 1H, J=7.1 Hz),1.36 (s, 9H), 1.56-1.68 (m, 1H), 2.03-2.11 (m, 2H), 2.21-2.31 (m, 1H),2.37-2.45 (m, 1H), 2.61-2.63 (m, 1H), 4.09-4.16 (m, 5H), 5.10 (bd, 1H,J=6.6 Hz), 7.11 (bs, 1H).

¹³C-NMR (75 MHz, CDCl₃): 13.9, 14.0, 17.7, 24.9 (d, J=1.0 Hz), 28.1,31.5 (d, J=10.9 Hz), 34.9 (d, J=9.4 Hz), 36.4 (d, J=8.8 Hz), 49.1, 61.5,61.8, 67.4, 77.2, 81.5 (d, J=242.6 Hz), 155.4, 168.6 (d, J=25.1 Hz),171.8 and 172.7 ppm.

Isomer B:(1S,2R,5S,6S,2′R)-Diethyl-2-(2′-tert-butoxycarbonylamino-propionylamino)-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylate

¹H-NMR (300 MHz, CDCl₃): 1.13 (t, 3H, J=7.1 Hz), 1.18 (t, 1H, J=7.1 Hz),1.31 (s, 9H), 1.51-1.60 (m, 1H), 1.98-2.07 (m, 2H), 2.15-2.22 (m, 1H),2.26-2.39 (m, 1H), 2.55-2.60 (m, 1H), 4.07-4.12 (m, 5H), 5.20 (sd, 1H),7.22 (bs, 1H).

¹³C-NMR (75 MHz, CDCl₃): 13.7, 13.8, 18.0, 24.7, 28.0, 31.4 (d, J=10.9Hz), 34.5 (d, J=8.9 Hz), 36.3 (d, J=9.4 Hz), 49.1, 61.4, 61.7, 67.3,77.2, 81.4 (d, J=242.6 Hz), 155.2, 168.5 (d, J=25.1 Hz), 171.7 and 172.8ppm.

Preparation 61(1R,2S,5R,6R)-2-Amino-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Reflux a solution of 30 mg (0.07 mmol) of Isomer A of Preparation 60 in6N HCl (2 mL) overnight. Remove the solvent under reduced pressure, washthe residue with ether, dissolve in MeOH (1 mL), and add propilene oxide(2 mL). Stir the mixture at room temperature overnight. Decant thesolvent, wash the residue with ether, and dry with an Ar stream to givethe title compound (12 mg, 85%) as a white solid.

[α]_(D) ²³=−25.0 (c=0.80 mg/mL, H₂O).

Preparation 62(1S,2R,5S,6S)-2-Amino-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Beginning with a solution Isomer B of Preparation 60, the title compoundis prepared essentially as Preparation 61.

[α]_(D)=21.7 (c=0.46 mg/mL, H₂O).

Preparation 63 Ethyl (6S)₄-thiabicyclo[3.1.0]hex-2-ene-6-carboxylate

A 3-neck, 5 liter flask is fitted with a stirrer, a thermocouple, and ateflon addition tube, and a N₂ inlet is charged 2000 ml of thiophene(d=1.05 g/mL, 25.0 mol at beginning and increasing to 45 mol withaddition of ethyl diazoacetate solution in thiophene, 17.1 equiv basedupon total addition of ethyl diazoacetate and not corrected for potencyof EDA, 19.0 equiv based upon potency corrected ethyl diazoacetate). Tothis is added, under N₂, 0.968 g of Rh₂(octanoate)₄ (1.24 mmoles, 0.0472mol % based upon g of ethyl diazoacetate not corrected for potency,0.052 mole % based upon moles of pure ethyl diazoacetate, JohnsonMathey: Lot No. 059255001). The suspension is heated to 46° C. andstirred at 46° C. for 10 minutes to affect dissolution to a greensolution. To the solution is added, by a positive displacement pump, asolution of 300 g of ethyl diazoacetate (90% pure, 2.63 moles, 2.37moles pura, 1.00 equiv, Aldrich: Lot No. 17603PI) dissolved in 1600 mLof thiophene. The rate of addition is such that the total addition timeis 8 hours; the slow rate of addition suppresses formation of themaleate and fumarate ethyl esters. When no ethyl diazoacetate remains(approximately thirty minutes), the dark amber reaction (3,985 g) iscooled to 23° C. The reaction mixture is split into portions and thelarger portion (3,240 g of 3,985 g total=81.3%) is directly concentratedto an oil. The crude product is passed through the wiped filmdistillation apparatus at 1.5 torr and 23° C. to degas the product andto remove residual amount of thiophene. The product is then distilled at120° C. and 1.3 torr. The title compound (faint yellow) is collected intwo fractions, 155.0 g and 32.2 g. Analysis by HPLC determines a potencyof 78% and 76% for the two lots respectively. Crystallization of thedistillate from above is accomplished by dissolution in methanol (2 mLper 1 g of distillate) and cooling to −10° C., at which time thesolution is optionally seeded if crystal growth is not observed. Oncecrystal growth has commenced, the mixture is further cooled to 45° C.and stirred for 2-3 hours, filtered, and washed with cold (−45° C.)methanol (1×1 mL per 1 g of distillate). The material is dried in vacuoat 24° C. to provide the title compound as a white to off-white solid in80-85% recovery and >98% potency.

Preparation 64 Ethyl(4S,6S)-4-hydroxy-2-thiabicyclo[3.1.0]hexane-6-carboxylate

To a solution of ethyl (6S)-4-thiabicyclo[3.1.0]hex-2-ene-6-carboxylate(22.2 g, 131 mmol) in 136 mL of tetrahydrofuran under nitrogen at 0° C.is added borane-THF complex (98 mL, 98 mmol) over 15-20 minutes. Afterstirring at 0° C. for 30 minutes, the reaction is allowed to warm to 15°C. and stir until complete by HPLC (1.5-2 hours). The reaction is cooledto 0° C. and transferred over 10-15 minutes to 111 mL of a pre-cooled(0° C.) 1 N pH 7 buffer solution while maintaining the temperature at 0°C. To the mixture is added sodium perborate monohydrate (15.6 g, 157mmol) as a solid in five portions such that the temperature ismaintained below 200C. The solution is allowed to warm to roomtemperature and stir for 1 hour followed by the addition of 222 mL ofwater. After stirring for 2 hours, the peroxides are quenched by addingsodium thiosulfate pentahydrate (9.7 g) dissolved in 24 mL of waterfollowed by stirring for 10 minutes. The mixture is extracted with ethylacetate (2×222 mL). The combined organic extracts are washed withsaturated aqueous sodium bicarbonate (1×222 mL) followed by brine (1×222mL) and concentration in vacuo to dryness. The crude product isdissolved in 1,2-dichloroethane (1 mL per 1 g of crude oil) and loadedonto a silica gel column (4 g of silica gel per 1 g of crude oilslurried and packed in 15% ethyl acetate-heptane). The column is elutedwith 15% ethyl acetate-heptane until product is visible by TLC, at whichtime the solvent is switched to 50% ethyl acetate-heptane. All fractionscontaining the title product are combined and concentrated in vacuo toan oil. Overall yield is in the 55-65% range.

Preparation 65 Ethyl (6S)₄-oxo-2-thiabicyclo[3.1.0]hexane-6-carboxylate

To a solution of dimethylsulfoxide (33.4 mL, 471 mmol) in 194 mL ofmethylene chloride at −70° C. is slowly added a solution oftrifluoracetic anhydride (33.2 mL, 235 mmol) in 73 mL of methylenechloride over 30 minutes (temperature maintained below −66° C.). Afterstirring for 20 min, a solution of ethyl(4S,6S)-4-hydroxy-2-thiabicyclo[3.1.0]hexane-6-carboxylate (34.1 g, 181mmol) in 194 mL of methylene chloride is added over 60 minutes such thatthe temperature is maintained below 60° C. After stirring for 1 hour,the reaction is treated with triethylamine (75.7 mL, 543 mmol) over 35minutes such that the temperature remains below −50° C. The reaction isallowed to stir an additional 1 hour, at which time the cooling bath isremoved and 400 mL of 2 N hydrochloric acid is added. Upon warming to 0°C., the layers are separated and the organic layer is washed with 2Nhydrochloric acid (1×300 mL), 1N aqueous sodium bicarbonate (1×670 mL),and water (1×300 mL) followed by drying over sodium sulfate, filtering,and concentrating in vacuo to a red oil which solidifies upon standing.The crude product is applied to a pad of silica gel (2 g per 1 g ofstarting alcohol packed with methylene chloride) and eluted withmethylene chloride (200-300 mL). All fractions containing product arecollected and concentrated to provide the title compound as anorange/brown solid. Typical corrected yields are in the 85-90% range.

Preparation 66(6S,11S)-8,10-dioxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid

Ammonium carbonate (2.46 g, 25.6 mmol) and potassium cyanide (0.817 mg,12.5 mmol) are combined in 19.9 mL of methanol and allowed to stir for30 minutes. The mixture is treated with a solution of ethyl(6S)-4-oxo-2-thiabicyclo[3.1.0]hexane-6-carboxylate (2.39 g, 12.8 mmol)in 19.9 mL of methanol and the reaction is heated to 30° C. and stirredfor 23 hours. The volatiles are evaporated and the residue is dissolvedin 2.75N sodium hydroxide (13.1 mL) and stirred for 1 hour. Afterdilution with 13.1 mL of water, the pH is lowered to 3.1 withconcentrated hydrochloric acid and optionally seeded with(6S,11R)-8,10-dioxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid. The pH is lowered to 1.0 and the suspension is cooled to 0° C. andstirred for 1.25 hours. The tan solid is collected, washed with coldwater (2.3 mL and 0.8 mL), and dried overnight in vacuo at 40° C. giving2.00 g (55% corrected for purity) of(6S,11R)-8,10-dioxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid. The filtrate is diluted with 50 mL of ethyl acetate and treatedwith 18 g of sodium chloride. After stirring for 15 minutes, the layersare separated and the aqueous layer is further washed with ethyl acetate(2×50 mL). The combined organic extracts are dried with sodium sulfate,filtered, and concentrated in vacuo to a slurry (ca. 5 mL) to which isadded tert-butyl methyl ether (25 mL) followed by stirring overnight.The solid is collected, washed with tert-butyl methyl ether, and driedin vacuo at 40° C. for 2 hours to provide 0.64 g (10% corrected forpurity) of the title compound as a 1:1 mixture of diastereomerichydantoins. This second crop of hydantoins is combined with the firstcrop and subjected to the next step.

Resolution

To a slurry of racemic acid (15 g, 65.7 mmol, ca. 6:1 ratio ofdiastereomeric hydantoins) in 300 mL of ethanol and 75 mL of water isadded (R)-phenylglycinol (9.0 g, 65.7 mmol). The mixture is heated toca. 80° C. to effect dissolution. The dark solution is allowed to coolslowly and precipitation was observed at 40-45° C. The slurry is furthercooled 0° C. and held for 1-1.5 hours. The solid is collected, washed(with stirring) with 4:1 ethanol:water (1×60 mL, pre-chilled to 0° C.),and dried in vacuo at 65° C. for 12-24 hours. Typical yields of theresolved salt are in the 37-45% range with >98% de and >98% ee observed.The resolved salt is dissolved in 6 volumes (mL per g) of water followedby treatment with 1.1 equivs. of conc HCl. The slurry is cooled to 0° C.and allowed to stir for 1 hour followed by filtration, rinsing with 1volume of cold water, and drying in vacuo at 60° C. Typical yields ofthe title compound are >90% with % de and % ee >99%.

Preparation 67(6S,11S)-2,2,8,10-Tetraoxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid

To a mixture of 6.8 mL of water, 0.7 mL of 50% aqueous sodium hydroxide,and 186 mg (0.74 mmol) of tungstic acid is added(6S,11S)-8,10-dioxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid (3.4 g, 14.9 mmol). The resulting solution is heated to 50° C. andtreated with 35% hydrogen peroxide (7.7 mL, 74.5 mmol) slowly over 66minutes. The reaction is allowed to stir thereafter at 47-48° C. for 5hours, followed by cooling to 0° C., filtering over a thin pad ofCelite, and rinsing with cold water (1×2 mL). The filtrate is heated to50° C. and treated with concentrated hydrochloric acid to pH=1.5. Theslurry is allowed to cool to room temperature and stir over night. Uponcooling to 0° C., the slurry is filtered, washed with cold water (2×2mL), and vacuum dried at 55° C. to a constant weight, providing 3.19 g(82%) of the title compound as a white solid:

[α]²⁵ _(D) −48.6 (c, 1.19, 1 N NaOH).

mp 275° C. (gray), 295° C. (brown).

500 MHz ¹H NMR (DMSO-d₆) 813.15 (br s, 1H), 10.99 (s, 1H), 8.13 (s, 1H),3.85 (d, 1H, J=15.0 Hz), 3.74 (dd, 1H, J=7.0, 4.0 Hz), 3.03 (d, 1H,J=15.5 Hz), 2.80 (dd, 1H, 7.0, 4.0 Hz), 2.39 (t, 1H, J=4.0 Hz).

¹³C NMR (125 MHz, DMSO-d₆) δ 174.39, 169.87, 156.35, 62.67, 52.59,44.16, 31.69, 21.92; FTIR (KBr) 3317 (s), 3250 (s), 3211 (s), 3086 (w),1791 (s), 1742 (s), 1713 (s), 1327 (s), 1192 (s), 1140 (s) cm⁻¹.

Anal. Cald for C₈H₈N₂O₆S: C, 36.93; H, 3.10; N, 10.77. Found: C, 36.76;H, 3.07; N, 10.60.

Preparation 68(1R,4S,5S,6S)-4-[(2′S)-(2′-Amino)-propionyl]amino-(2-sulfonylbicyclo[3.1.0]hexane)-4,6-dicarboxylicacid

To a stainless steel Parr reactor is added(6S,11S)-2,2,8,10-Tetraoxo-2-thiaspiro[bicyclo[3.1.0]hexane-4,5′-imidazolidine]-6-carboxylicacid (2.50 g, 9.60 mmol) and 2N sodium hydroxide (24.0 mL, 48.0 mmol).After the mixture is heated to 95° C. and stirred for 21 hours, themixture is cooled to room temperature and treated with activatedcharcoal (1.25 g). The mixture is filtered through Celite and thefiltrate is concentrated to 17 g and diluted with H₂O to afford a weightof 24 g. The pH is lowered to 6.5 using conc. HCl and the mixture heatedto 62° C. After the pH is lowered to 2.5 using conc. HCl,crystallization occurred. The suspension is allowed to cool to 30° C.before the pH is adjusted to 1.7 and its temperature lowered to 5° C.After the suspension is held at this temperature for 18 hours, the solidis collected and washed with cold H₂O (2×2.9 mL). The white solid isdried in vacuo at 45° C. to afford the title compound (1.81 g, 80%). Thetitle compound is slurried in 10 volumes of water and heated to 85° C.for 3-4 hours, cooled to room temperature, stirred for 2-3 hours,filtered, and washed with water (1×1 volume). Recovery is >95%.

Preparation 69 Ethyl (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate

To a suspension of (ethoxycarbonylmethyl)dimethyl sulfonium bromide (134g, 585 mmol) in 486 mL of acetonitrile at room temperature is added 87.4mL (585 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene dropwise over 15minutes. After stirring for 1 hour, the yellow mixture is treated with40 g (487 mmol) of 2-cyclopenten-1-one over 10 minutes. The mixture isallowed to stir over night at which time 480 mL of tert-butyl methylether is added, followed by washing with 1N hydrochloric acid (1×240mL). The aqueous layer was washed with tert-butyl methyl ether (1×240mL). The combined organic extracts were washed with brine (1×400 mL),dried (MgSO₄), filtered, and concentrated in vacuo to provide crudeethyl (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate as an orange solid(84.8 g). The crude material may be purified through distillation (138°C., 10 mm Hg), followed by slurrying the solidified distillate inheptane, filtering, and drying.

Preparation 70 (±)(6S)-2-Oxobicyclo[3.1.0]hexane-6-carboxylic acid

To a solution of crude ethyl(6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate (30.2 g, 180 mmol,uncorrected) in 30 mL of ethanol at room temperature is added 89 mL (178mmol) of 2N sodium hydroxide. Upon stirring for 80 minutes, the reactionmixture is washed with tert-butyl methyl ether (1×90 mL) and the aqueouslayer is treated with conc. hydrochloric acid (18 mL) to reach a pH=1.0.The mixture is treated with 15 g of sodium chloride followed by washingwith ethyl acetate (3×90 mL). The combined organic extracts are dried(Na₂SO₄), filtered, and concentrated in vacuo to give 23.8 g (94%,uncorrected) of the title compound as an off-white solid.

Preparation 71 (+) (6S)-2-Oxobicyclo[3.1.0]hexane-6-carboxylic acidN-benzyl-α-methylbenzylamine salt

To a solution of crude (±) (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylicacid (11.9 g, 84.9 mmol, assume 100% potency) in 119 mL of 6:1 ethylacetate:ethanol at reflux is added 18 g (85.1 mmol) of(S)-N-benzyl-α-methylbenzyl-amine. Upon dissolution, the mixture isallowed to cool optionally followed by seeding at 52° C. Upon cooling toroom temperature and stirring an additional 13.5 b, the crystals arecollected and washed with 6:1 ethyl acetate:ethanol (2×48 mL). Drying invacuo gave 10.8 g (36%, 77% de) of the resolved salt as a solid.

The de of the salt is determined by chiral GC analysis of the derivedmethyl ester prepared as follows: 150 mg of the resolved salt isdissolved in 5 mL of methylene chloride and is washed with 1 N sulfuricacid (2×1 mL). The organic layer is dried, filtered, diluted with 2 mLof methanol, and treated with 1 mL of 2 M trimethylsilyl diazomethane inhexanes. After stirring at room temperature for 15 minutes, the mixtureis concentrated in vacuo to provide the methyl ester suitable for chiralGC analysis. GC conditions: 30 m×0.25 mm×0.25 ti β-DEX 325 column, 140°C. oven temperature, helium carrier gas @ 1 mL/min, FID detection at250° C., 1 μl split 1:100, sample @ 1 mg/mL in methylene chloride.

Preparation 72 Ethyl (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate

To a suspension of 46.3 g (132 mmol) of(6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylic acidN-benzyl-α-methylbenzylamine salt in 200 mL of ethyl acetate is added198 mL (198 mmol) of 2N sodium hydroxide. After mixing well, the layersare separated and the aqueous layer is washed with ethyl acetate (1×200mL). The aqueous layer is treated with 18 mL (211 mmol) of conc.hydrochloric acid and 100 g of sodium chloride. The mixture is allowedto stir for 30 minutes followed by washing with ethyl acetate (2×200mL). The combined organics are dried (MgSO₄), filtered, and concentratedin vacuo to provide 18.3 g (99%) of the resolved acid [(+)(6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylic acid] as a white solid.

Next, 10 g (71 mmol) of crude resolved acid product from above isdissolved in 42 mL of ethanol and treated with 4 mL (71 mmol) of conc.sulfuric acid dropwise. The mixture is heated to 45° C. and is allowedto stir for 75 minutes. Upon cooling to room temperature, 42 mL of wateris added along with 20 mL of ethyl acetate and 12 g of sodiumbicarbonate. Upon stirring for several minutes, the mixture is washedwith ethyl acetate (2×50 mL). The combined organics are dried (MgSO₄),filtered, and concentrated in vacuo to provide 11 g (92%) of crude ethyl(6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate as a white solid.Crystallization from 6:1/heptane:tert-butyl methyl ether (3.5 mL per gof substrate) provided this title compound in approximately 80% yieldand >98% ee as determined by chiral GC analysis.

Preparation 73 (6S)-6-(Ethoxycarbonyl)bicyclo[3.1.0]hex-2-en-2-ylacetate

A mixture of ethyl (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate (380.1g, 2.26 mol) and sulfuric acid (18 M, 6.3 mL, 0.11 mol) in isopropenylacetate (2.26 L) is heated at reflux using a Dean-Stark apparatus for2.5 hours, at which time GC analysis revealed a 9:1 mixture of the titlecompound versus ethyl (6S)-2-oxobicyclo[3.1.0]hexane-6-carboxylate.After the removal of 950 mL of solvent by distillation over 1 hour, GCshows that the product/starting material ratio is 17:1. Additionalisopropenyl acetate (900 mL) and conc. H₂SO₄ (3.15 mL) are added, andthe mixture is stirred at reflux for another 15 hours, at which time GCshows 27:1 product/starting material. After another 1.35 L of solvent isdistilled off, the mixture is cooled to room temperature before it isdiluted with MTBE (2 L), H₂O (250 mL), and aqueous saturated NaHCO₃ (600mL). The layers are separated and the organic layer is washed with brine(400 mL). The combined aqueous layers are extracted with MTBE (400 mL),and the combined organic layers are dried (Na₂SO₄), filtered, andconcentrated to a dark red/brown oil (540 g). The crude oil is splitinto two equal portions and filtered through a pad of flash SiO₂ (713 gfor each batch), eluting with 10:1/heptane:ethyl acetate. Theproduct-containing fractions from both plugs are combined andconcentrated to afford the title compound as a yellow oil (460 g, 97%;90% corrected for solvent by NMR). Column chromatography on silica geleluting with ethyl acetate/hexanes (1:5) provides an analytically puresample of the title compound as a colorless oil.

[α]²⁵ _(D) +185 (c 1.48, CHCl₃).

500 MHz ¹H NMR (CDCl₃) δ 5.19-5.18 (m, 1H), 4.12 (q, 1H, J=7.0 Hz), 4.11(q, 1H, J=7.0 Hz), 2.74-2.69 (m, 1H), 2.48-2.43 (m, 2H), 2.22-2.19 (m,1H), 2.16 (s, 3H), 1.39 (dd, 1H, J=2.5, 2.5 Hz), 1.25 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 173.37, 169.01, 152.26, 111.56, 61.28, 32.47,32.40, 29.72, 24.97, 21.67, 14.95.

FTIR (CHCl₃) 3026 (m), 2985 (m), 1724 (s), 1272 (s), 1187 (s) cm⁻¹.

ES HRMS calcd for C, H₁₈NO₄ [M+NH₄]+228.1236, found 228.1252.

Preparation 74 Ethyl(3S,1R,6R)-7-oxa-5-oxotricyclo[4.1.0.0<2,4>]heptane-3-carboxylate

A mixture of (6S)-6-(ethoxycarbonyl)bicyclo[3.1.0]hex-2-en-2-yl acetate(212.2 g, 1.01 mol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (252.0g, 1.11 mol) in 2.02 L of 1,4-dioxane is heated to reflux and stirredfor 17 hours, at which time GC analysis shows complete conversion toethyl (6S)-4-oxobicyclo[3.1.0]hex-2-ene-6-carboxylate. The mixture iscooled to room temperature and diluted with THF (564 mL). After themixture is cooled to 8° C., 1,8-diazabicyclo[5.4.0]undec-7-ene (377 mL,2.52 mol) is added over 30 minutes such that the reaction temperature ismaintained below 10C. The mixture is then cooled to 5° C., andtert-butyl hydroperoxide (70 wt % in water, 210 mL, 1.51 mol) is addedover 50 minutes, maintaining the reaction temperature below 9° C. Afterthe mixture stirred another 50 minutes, the reaction is filtered and thebrown cake is washed with MTBE (2×800 mL). To the filtrate is added 1.20L of 1N HCl and, after mixing well, the layers are separated. Theorganic layer is washed sequentially with aqueous saturated NaHCO₃ (1.20L), aqueous saturated Na₂S₂O₃ (1.20 L), and brine (600 mL). After thesolution is dried (Na₂SO₄), it is concentrated to an orange sludge whichis diluted with 200 mL of heptane. The volatiles are evaporated toproduce an orange solid that is triturated with 350 mL of heptane andfiltered, washing the cake with additional heptane (2×175 mL). Thecollected solid is dried in vacuo at room temperature for 17 hours toprovide 138.7 g (75%) of the title compound as a brown-yellow solid.Crystallization from MTBE provides an analytically pure sample of thetitle compound as a white solid.

[α]²⁵ _(D) +2.3 (c 1.20, CHCl₃), +8.4° (c 1.28, acetone); mp 129-130° C.

500 MHz ¹H NMR (CDCl₃) δ 4.16 (q, 2H, J=7.0 Hz), 3.99 (t, 1H, J=2.5 Hz),3.24-3.23 (m, 1H), 2.96-2.94 (m, 1H), 2.21-2.19, (m, 1H), 2.08 (t, 1H,J=3.0 Hz), 1.26 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 201.19, 168.84, 62.42, 57.04, 51.25, 31.16,30.54, 29.60, 14.79.

FTIR (KBr) 3087 (w), 3059 (w), 3051 (w), 3007 (w), 2993 (w), 2963 (w),1753 (s), 1719 (s), 1273 9s), 1191 (s), 1009 (m), 848 (m) cm⁻¹.

Anal. Cald for C₉H₁₀O₄: C, 59.34; H, 5.53. Found: C, 59.32; H, 5.43.

Preparation 75 Ethyl (6S)-4-oxobicyclo[3.1.0]hex-2-ene-6-carboxylate

Although the title compound is typically used in situ in the preparationof ethyl(3S,1R,6R)-7-oxa-5-oxotricyclo[4.1.0.0<2,4>]heptane-3-carboxylate, ananalytically pure sample of the title compound is obtained by filteringthe reaction mixture containing this compound and evaporating thefiltrate to give a brown solid. The solid is resuspended in ethylacetate, the suspension filtered, and the filtrate concentrated.Chromatography of the residue on silica gel with ethyl acetate/hexanes(1:5 to 1:2) gives the title compound, which is recrystallized from hotethyl acetate and chromatographed again using the previous conditions togive the title compound as a white solid.

[α]²⁵ _(D) −268 (c 1.17, CHCl₃).

mp 97-98° C.

500 MHz ¹H NMR (CDCl₃) δ 7.60 (ddd, 1H, J=5.5, 2.5, 0.75 Hz), 5.73 (dd,1H, J=5.0, 0.5 Hz), 4.15 (q, 2H, J=7.0 Hz), 2.96-2.94 (m, 1H), 2.63-2.61(m, 1H), 2.60 (t, 1H, J=2.5 Hz), 1.26 (t, 3H, 17.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 203.96, 168.61, 160.33, 130.29, 62.03, 46.53,30.72, 29.62, 14.82.

FTIR (KBr) 3080 (m), 2996 (m), 1717 (s), 1695 (s), 1266 (s), 1291 (m),1191 (s), 11179 (s) cm⁻¹.

Anal. Cald for C₉H₁₀O₃: C, 65.05; H, 6.07. Found: C, 64.97; H, 6.01.

Preparation 76 Ethyl(4S,6S)₄-hydroxy-2-oxobicyclo[3.1.0]hexane-6-carboxylate

A stirred solution of ethyl(3S,1R,6R)-7-oxa-5-oxotricyclo[4.1.0.0<2,4>]heptane-3-carboxylate (36.3g, 0.20 mol) in 667 mL of acetone is treated sequentially with sodiumacetate (36.1 g, 0.44 mol), sodium iodide (65.8 g, 0.44 mol), and aceticacid (27.5 mL, 0.48 mol). The mixture is allowed to stir at 30° C. for15 hours before the acetone is removed in vacuo leaving behind a brownsolid that is partitioned between ethyl acetate (323 mL) and H₂O (323mL). The layers are separated and the aqueous layer is washed with ethylacetate (3×323 mL). The combined organics are washed sequentially withaqueous saturated Na₂S₂O₃ (364 mL) and aqueous saturated NaHCO₃ (364mL). Each aqueous wash is back-extracted with ethyl acetate (323 mL).The combined organics are dried (Na₂SO₄), filtered, and concentrated toa red-brown oil which was dissolved in 300 mL of ethanol. Evaporation ofthe volatiles affords the title product as a red-brown oil (41.8 g,114%). Column chromatography on silica gel using ethyl acetate/hexanes(1:2 to 2:1) followed by crystallization from hot MTBE provides ananalytically pure sample of the title compound as a white solid.

[α]²⁵ _(D) +3.9 (c 1.39, CHCl₃), +6.0° (c 1.69, MeOH).

mp 81-82° C.

500 MHz ¹H NMR (CDCl₃) δ 4.60 (br s, 1H), 4.16 (q, 2H, J=7.0 Hz), 2.66(dd, 1H, J=5.0, 4.0 Hz), 2.42-2.40 (m, 1H), 2.34 (dd, 1H, J=19.0, 5.5Hz), 2.24, (br d, 1H, J=3.0 Hz), 2.07 (d, 1H, J=19.0 Hz), 1.91 (t, 1H,J=3.0 Hz), 1.27 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 209.74, 170.07, 69.04, 62.32, 43.47, 36.89,34.95, 26.14, 14.83.

FTIR (CHCl₃) 3607 (w), 3447 (w), 3025 (m), 2985 (w), 1739 (s), 1728 (s),1270 (s). 1187 (s) cm⁻¹.

Anal. Cald for C₉H₁₂O₄: C, 58.69; H, 6.57. Found: C, 58.48; H, 6.63.

Preparation 77 Ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-cyano-4-hydroxybicyclo[3.1.0]hexane-6-carboxylate

To a solution of ethyl(4S,6S)-4-hydroxy-2-oxobicyclo[3.1.0]hexane-6-carboxylate (68.2 gcorrected to 60.0 g due to ethanol contamination, 0.326 mol) in ethanol(332 mL) and H₂O (332 mL) is added (R)-methylbenzylamine (46.3 mL, 0.359mol) and NaCN (20.8 g, 0.424 mol), maintaining the temperature between20 and 25° C. Conc. HCl (35.3 mL, 0.424 mol) is then added over 10 minwhile maintaining the above reaction temperature. The dark brown mixtureis stirred for 1 hour before it is optionally seeded with the titlecompound to initiate crystallization. The suspension is stirred for 1hour before H₂O (664 mL) is added. After the suspension stirs another1.75 hours, the title compound is collected as a tan solid which iswashed with H₂O (332 mL). Air is pulled through the wetcake on thefilter for 25 minutes before the material is used directly in thenitrile hydrolysis (wetcake weight 145 g). Although the title compoundquickly decomposes during in vacuo drying at temperatures greater than25° C., it is possible to dry small samples in vacuo at room temperaturewithout decomposition.

[α]²⁵ _(D) +81.6 (c 1.18, CHCl₃).

mp 70-720C (decomp).

500 MHz ¹H NMR (CDCl₃) δ 7.39 (d, 2H, J=7.0 Hz), 7.26-7.16 (m, 3H), 4.31(d, 1H, J==5.0 Hz), 4.22 (q, 1H, J=6.5 Hz), 3.93-3.85 (m, 2H), 2.33 (d,1H, J=15.0 Hz), 2.01 (br t, 1H, J=4.5 Hz), 1.64 (dd, 1H, J=15.0, 5.0Hz), 1.55-1.54 (m, 1H), 1.40-1.39 (m, 4H), 1.17 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 170.54, 144.85, 128.61, 127.45, 127.38,121.88, 72.17, 61.02, 60.66, 56.57, 45.82, 36.70, 34.45, 25.83, 21.75,14.22.

FTIR (KBr) 3568 (m), 3489 (m), 3285 (m), 2923 (m), 2228 (w), 1712 (s),1298 (m). 1197 (m) cm⁻¹.

FAB HRMS calcd for C₁₈H₂₃N₂O₃ [M+H]⁺ 315.1709, found 315.1704.

Preparation 782-[((1R)-1-Phenylethyl)amino](2S,4S,6R)-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid

To a solution of ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-cyano-4-hydroxybicyclo[3.1.0]hexane-6-carboxylatewetcake (0.326 mmol theory) in DMSO (220 mL) is slowly added 30% H₂O₂(44.5 mL, 0.426 mol), maintaining the temperature below 27° C. Thetemperature is lowered to 19° C. and 5N NaOH (52.3 mL, 0.262 mol) iscarefully and slowly added at first over 15 minutes, maintaining thetemperature between 22 and 27° C. An ice bath of appropriate capacity isrequired to handle the exotherm of this reaction. After the brown,heterogeneous mixture is stirred for 20 minutes at the above temperaturerange, HPLC showed that the starting material had been consumed to givean amide intermediate. After the reaction is stirred another 1.5 hours,Na₂SO₃ (13.7 g, 0.109 mol) is added and the mixture stirs for 15minutes, at which time the mixture tests negatively for peroxides bystarch-iodide paper. Following the addition of 3N NaOH (291 mL, 0.873mol), the mixture is heated to 85° C. and stirred for 18 hours. Thehomogeneous brown mixture is cooled to 30° C. and conc. HCl is added tolower the pH to 3.6 while maintaining the temperature between 30 and 35°C. After crystallization begins at pH 3.6, the suspension is stirred for15 minutes before the pH is lowered to 2.5. After the mixture is stirredfor 10 additional minutes, it is cooled to 2° C. and stirred for 2 hoursbefore the gray solid is collected and washed with cold H₂O (400 mL) andEtOH (300 mL). The collected solid is dried in vacuo at 45° C. for 17hours to provide 42.9 g (43% from the start of Preparation 18) of thetitle compound. In order to forward process all of the title compoundproduced in the reaction, it is recovered from the mother liquor in thefollowing manner. The ethanol portion of the mother liquor is evaporatedand the residue is combined with the aqueous portion of the motherliquor. Following the distillation of H₂O (485 mL) under reducedpressure, the pH of the mother liquor is adjusted to 12.9 with 70 mL of5N NaOH and 5 mL of 50% NaOH. After the solution is washed with n-BuOH(3×800 mL), its pH is adjusted to 2.5 with conc. HCl and the solution isconcentrated. The residue is diluted with EtOH (100 mL) and thevolatiles evaporated (2×). The residue is diluted with EtOH (150 mL) andthe tan solid containing additional title compound and salts is washedwith EtOH (75 mL) and dried at 50° C. in vacuo to a weight of 102 g.Both crops of the title compound were used in the subsequentesterification.

[α]²⁵ _(D) +4.5 (c 1.41, 1 N NaOH).

mp 220° C. (gray from off-white), 280° C. (brown).

500 MHz ¹H NMR (D₂O, KOD) δ 7.39 (d, 2H, J=7.0 Hz), 7.19-7.04 (m, 5H),3.92 (d, 1H, J=5.0 Hz), 3.67 (q, 1H, J=7.0 Hz), 1.76 (d, 1H, J=15.0 Hz),1.54-152 (m, 1H), 1.37 (dd, 1H, J=15.0, 5.0 Hz), 1.15 (d, 3H, J=6.5 Hz),1.12 (dd, 1H, J=6.0, 3.0 Hz), 0.92 (t, 1H, J=3.3 Hz).

¹³C NMR (125 MHz, D₂O, KOD) δ 185.82, 182.96, 148.01, 131.31, 129.97,129.78, 74.99, 73.84, 58.78, 46.91, 38.05, 35.02, 27.34, 27.15.

FTIR (KBr) 3366 (m), 3072 (s), 2886 (s), 1696 (m), 1611 (m), 1560 (m),1455 (m), 1377 (m), 1278 (m), 1202 (m), 1188 (m) cm⁻¹.

Anal. Cald for C₁₆H₁₉NO₅: C, 62.94; H, 6.27; N, 4.59. Found: C, 62.70;H, 6.21; N, 4.67.

Preparation 79 Ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-carbamoyl-4-hydroxybicyclo[3.1.0]hexane-6-carboxylate

Although the title compound is typically used in situ in the preparationof2-[((1R)-1-phenylethyl)amino](2S,4S,6R)₄-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid, the compound could be isolated albeit with some yield loss due toaccompanying ester hydrolysis during the nitrile hydrolysis. In theisolation, the nitrile hydrolysis reaction mixture is partitionedbetween CH₂Cl₂ and H₂O as soon as ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-cyano-4-hydroxybicyclo[3.1.0]hexane-6-carboxylateis consumed. After the organic layer is dried (MgSO₄) and concentrated,the residue is purified by silica gel column chromatography usingEtOAc/hexanes (2:1) to EtOAc to afford the title compound as a whitefoam.

[α]²⁵ _(D) +61.3 (c 1.20, CHCl₃).

500 MHz ¹H NMR (CDCl₃) δ 7.32-7.20 (m, 5H), 7.19 (br d, 1H, J=4.0 Hz),5.49 (br d, 1H, J=4.0 Hz), 4.88 (d, 1H, J=11.5 Hz), 4.24 (dd, 1H,J=11.5, 6.0 Hz), 4.06-4.00 (m, 2H), 3.77 (q, 1H, J=7.0 Hz), 2.21 (d, 1H,J=15.0 Hz), 2.18-2.15 (m, 2H), 1.71 (br s, 1H), 1.54 (dd, 1H, J=14.5,6.0 Hz), 1.38, (d, 3H, J=6.5 Hz), 1.32 (t, 1H, J=3.3 Hz), 1.24 (t, 3H,J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 180.42, 171.47, 146.05, 128.97, 127.43,126.48, 73.16, 70.76, 61.08, 56.00, 42.82, 35.97, 35.67, 26.13, 21.53,14.34.

FTIR (CHCl₃) 3441 (m), 3345 (m), 2975 (w), 1725 (s), 1665 (s), 1288,1186 (m) cm⁻¹.

Anal. Cald for C₁₈H₂₄N₂O₄: C, 65.04; H, 7.28; N, 8.43. Found: C, 65.41;H, 7.58; N, 8.32.

Preparation 80 Ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-(ethoxycarbonyl)₄-hydroxybicyclo[3.1.0]hexane-6-carboxylate

To a suspension of2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylicacid (4 g, 13 mmol) in 48 mL of ethanol at room temperature is addedacetyl chloride (11.2 mL, 157 mmol) via an addition funnel such that agentle reflux is maintained. The resulting mixture is allowed to stiranother 16 hours at reflux and upon cooling to room temperature isconcentrated in vacuo to a solid residue. The solid is treated slowlywith a solution of sodium bicarbonate (6.6 g) in 100 mL of waterfollowed by washing with ethyl acetate (2×100 mL). The combined organicsare dried (MgSO₄), filtered, and concentrated in vacuo to give 4.7 g(99%) of the title compound as a solid. Column chromatography on silicagel eluting with CH₂Cl₂/MeOH (95:5) followed by crystallization fromEt₂O provides an analytically pure sample of the title compound as awhite solid.

[α]²⁵ _(D) +52.5 (c 1.30, CHCl₃).

mp 73-74° C.

500 MHz ¹H NMR (CDCl₃) δ 7.29-7.14 (m, 5H), 4.25 (dq, 1H, 11.0, 7.0 Hz),4.18 (dd, 1H, J=9.5, 5.5 Hz), 4.10 (dq, 1H, J=11.0, 7.0 Hz), 3.92 (dq,1H, J=11.0, 7.0 Hz) 3.82 (dq, 1H, J=11.0 Hz, 7.0 Hz), 3.67 (q, 1H, J=7.0Hz), 2.73 (d, 1H, J=9.5 Hz), 2.15-2.12 (m, 2H), 2.01-1.99 (m, 1H), 1.89(dd, 1H, J=6.0, 3.0 Hz), 1.61 (dd, 1H, J=15.0, 6.0 Hz), 1.36 (t, 1H,J=3.5 Hz), 1.33-1.30 (m, 6H), 1.18 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 178.11, 171.59, 146.32, 128.41, 127.07,126.85, 73.33, 70.15, 62.07, 60.75, 56.66, 44.72, 36.78, 33.61, 26.24,20.07, 14.37, 14.23.

FTIR (KBr) 3492 (s), 3303 (m), 3055 (w), 2981 (w), 2896 (w), 1722 (s),1705 (s), 1289 (m), 1251 (m), 1177 (m) cm⁻¹.

Anal. Cald for C₂₀H₂₇NO₅: C, 66.46; H, 7.52; N, 3.88. Found: C, 66.42;H, 7.44; N, 3.92.

Preparation 81 Ethyl2-[((1R)-1-phenylethyl)amino](2S,4R,6R)-2-(ethoxycarbonyl)₄-fluorobicyclo[3.1.0]hexane-6-carboxylate

To a solution of ethyl2-[((1R)-1-phenylethyl)amino](2S,4S,6R)-2-(ethoxycarbonyl)-4-hydroxybicyclo[3.1.0]hexane-6-carboxylate(59.0 g crude, 0.163 mol) in CH₂Cl₂ (690 mL) at −20° C. is addedDeoxo-Fluor® (45.1 mL, 0.245 mol) over 15 minutes, maintaining thetemperature between −15 and −20° C. The mixture is stirred for 20minutes at this temperature and at 0° C. for 15 minutes before aqueous15% Na₂CO₃ (650 ml) is slowly added while maintaining the temperaturebelow 10° C. The layers are separated and the aqueous layer backextracted with CH₂Cl₂ (150 mL). The combined organic layers are dried(Na₂SO₄) and concentrated to a brown oil (73 g). The oil is purified ona pad of silica gel (400 g) eluting with EtOAc/heptane (1:6) to affordthe title compound as a yellow oil (49.7 g, 84%).

[α]²⁵ _(D) +36.2 (c 1.30, CHCl₃).

500 MHz ¹H NMR (CDCl₃) δ 7.29-7.14 (m, 5H), 5.22 (ddt, 1H, J=8.0, 4.5Hz, J_(HF)=56.0 Hz), 4.16 (dq, 1H, J=11.0, 7.0 Hz), 4.05 (dq, 1H, 11.0,7.0 Hz), 3.96 (dq, 1H, 10.5, 7.0 Hz), 3.85 (dq, 10.5, 7.0 Hz), 3.66 (q,1H, 6.5 Hz), 2.45 (dd, 1H, J=14.0, 8.0 Hz), 2.16-2.12 (m, 1H), 1.95 (t,1H, J=3.5 Hz), 1.81 (dt, 1H, J=3.5 Hz, J_(HF)=3.5 Hz), 1.51 (ddd, 1H,J=14.0, 8.0 Hz, J_(HF)=22.0 Hz), 1.32 (d, 3H, J=6.5 Hz), 1.27 (t, 3H,J=7.0 Hz), 1.21 (t, 3H, J=7.0 Hz).

¹³C NMR (125 MHz, CDCl₃) δ 175.29, 171.66, 146.21, 128.45, 127.03,126.90, 92.65 (d, J_(CF)=182 Hz), 68.68 (d, J_(CF)=4.9 Hz), 61.70,60.92, 56.13, 38.60 (d, J_(CF)=23.0 Hz), 33.07 (d, J_(CF)=7.6 Hz), 32.23(d, J_(CF)=22.0 Hz), 26.26, 20.22 (d, J_(CF)=3.9 Hz), 14.41, 14.24.

FTIR (CHCl₃) 3028 (w), 2983 (w), 1724 (s), 1705 (s), 1293 (m), 1242 (m),1190 (m), 1037 (m), 1013 (m) cm⁻¹.

Anal. Cald for C₂₀H₂₆FNO₄: C, 66.10; H, 7.21; N, 3.85. Found: C, 66.02;H, 7.00; N, 3.95.

Preparation 821R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

A mixture of ethyl2-[((1R)-1-phenylethyl)amino](2S,4R,6R)-2-(ethoxycarbonyl)-4-fluorobicyclo[3.1.0]hexane-6-carboxylate(68.4 g, 0.188 mol), conc. HCl (15.7 mL, 0.188 mol), and 10% Pd/C (dry,13.7 g) in EtOH (400 mL) is placed under hydrogen (50 psi) for 18 hours.The catalyst is filtered off and the filtrate is evaporated to give thetitle compound as an off-white foam (59.2 g, 106% corrected to 97% dueto EtOH contamination). Crystallization from EtOAc/MTBE provides ananalytically pure sample of the title compound as a white solid.

[α]²⁵ _(D) +^(55.6) (c 1.17, CHCl₃).

mp 86-88° C.

500 MHz ¹H NMR (CDCl₃) δ 9.20 (br s, 2H), 5.50 (ddt, 1H, J=8.0, 4.5 Hz,J_(HF)=56.0 Hz), 4.31 (q, 1H, J=7.0 Hz), 4.20-4.07 (m, 3H), 2.88 (t, 1H,J=3.0 Hz), 2.71 (dd, 1H, J=14.5, 8.0 Hz), 2.48-2.43 (m, 2H), 2.16 (ddd,1H, J=14.5, 7.5 Hz, J_(HF)=22.0 Hz), 1.34 (t, 3H, J=7.0 Hz), 1.25 (t,3H, J=7.0 Hz)

¹³C NMR (125 MHz, CDCl₃) δ 171.12, 169.41, 91.94 (d, J_(CF)=189 Hz),63.85, 63.66 (d, J_(CF)=3.8 Hz), 61.73, 34.55 (d, J_(CF)=26.4 Hz), 31.58(d, J_(CF)=7.8 Hz), 30.80 (d, J_(CF)=24.1 Hz), 20.22, 14.31, 14.21.

FTIR (KBr) 3353 (m), 3173 (w), 2745 (m), 1729 (s), 1547 (m), 1294 (m),1269 (m), 1195 (m), 1011 (m) cm⁻¹.

Anal. Cald for C₁₂H₁₈FNO₄: C, 48.74; H, 6.48; N, 4.74. Found: C, 48.80;H, 6.41; N, 4.76.

Preparation 831R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid

A solution of 3N NaOH (251 mL, 0.753 mol) is slowly added to1R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride (59.2 g crude, 0.188 mol theory), maintaining thetemperature below 26° C. After the mixture is stirred for 10 minutes, itis homogeneous. The mixture is stirred for 1.25 hours at roomtemperature before the pH is slowly lowered to pH 2.8 using conc. HClwhile maintaining the temperature between 20 and 26° C. At pH 2.8, themixture begins crystallizing, and the suspension is stirred at this pHfor 10 minutes before the pH is lowered to 2.1 with conc. HCl. Afteranother 15 minutes of stirring, i-PrOH (67 mL) is added and thesuspension is cooled to 0° C. and stirred for 2 hours. The solid iscollected and washed with 37 mL of cold H₂O/i-PrOH (4:1). The collectedsolid is dried in vacuo at 40° C. for 18 hours, affording the titlecompound as a white solid (33.1 g, 87% from the start of Preparation23).

Preparation 84 Reslurry of1R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid

A stirred suspension of1R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (33.0 g, 0.162 mmol) in H₂O (165 mL) is warmed to 89° C. over 1hour, and i-PrOH (41 mL) is added. The mixture is then stirred for 5minutes at reflux (83° C.) before it is allowed to cool to roomtemperature and stir for 4 hours. The product is collected, washed withi-PrOH/H₂O (1:4, 40 mL) and i-PrOH (25 mL), and dried in vacuo at 40° C.for 18 hours to afford the title compound as a white solid (30.6 g,93%).

Preparation 851R,2S,4R,5R,6R-2-Amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid ethyl ester

To a slurry of1R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (14.45 g, 71.12 mmol) in 202 mL of absolute ethanol at roomtemperature is added thionyl chloride (26 mL, 356 mmol) dropwise over 20minutes. The slurry is heated to reflux and allowed to stir for 3 hoursfollowed by cooling to room temperature overnight. The resultantsolution is concentrated in vacuo to a residue that is diluted with 136mL of ethyl acetate and treated with 306 mL of 10% aqueous sodiumcarbonate over 15 minutes with swirling by hand such that the final pHis 10. The layers are separated and the aqueous layer is washed withethyl acetate (1×136 mL). The combined organic extracts are washed withbrine (1×136 mL), dried (MgSO₄), filtered, and concentrated in vacuo toprovide 17.07 g (93%) of the title compound as white solid.

FDMS: M⁺+]=260.

Anal. calcd. For C₁₂H₁₈FNO₄.0.1H₂O: C, 55.21; H, 7.03; N, 5.37. Found:C, 55.10; H, 6.96; N, 5.22.

m.p.=64-66° C.

[α]_(D) ²⁵=+2° (c=0.96, MeOH), [α]_(D) ²⁵=+15 (c=1.21, DMSO)

Preparation 861R,2S,4R,5R,6R-2-[2′S-2′-(tert-butoxycarbonylamino)propionyl]amino-4-flurobicyclo[3.1.0]hexane-2,6-dicarboxylicacid ethyl ester

To a solution of N-Boc-L-alanine (38.62 g, 204 mmol) in 396 mL ofmethylene chloride at −22° C. under nitrogen is added N-methylmorpholine (22.44 mL, 204 mmol) followed by iso-butyl chloroformate(26.48 mL, 204 mmol) dropwise over 15 min such that the reactiontemperature did not exceed −18° C. The resultant thin slurry is allowedto stir at −20° C. for 30 minutes at which time a solution of1R,2S,4R,5R,6R-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid ethyl ester (49.46 g, 191 mmol) in 247 mL of methylene chloride isadded over 40 min such that the reaction temperature did not exceed −16°C. Upon completion of the addition, the reaction is removed from thecooling bath and is allowed to stir at ambient temperature for 70minutes at which time the reaction temperature had reached 15° C. andthe color became faint orange. The reaction is treated with 408 mL of 1N hydrochloric acid followed by stirring for 5 minutes and separation ofthe layers. The organic layer is washed with saturated aqueous sodiumbicarbonate (1×408 mL), dried (Na₂SO₄), filtered, and concentrated invacuo to a white foam (88.16 g).

FDMS: M⁺+1=260.

Anal. calcd. For C₁₂H₁₈FNO₄.0.1H₂O: C, 55.21; H, 7.03; N, 5.37. Found:C, 55.10; H, 6.96; N, 5.22.

m.p.=64-66° C.

[α]_(D) ²⁵=+2° (c=0.96, MeOH), [α]_(D) ²⁵=+15 (c=1.21, DMSO).

Preparation 871R,2S,4R,5R,6R-2-[2′S-2′-(tert-butoxycarbonylamino)propionyl]amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid

To a solution of1R,2S,4R,5R,6R-2-[2′S-2′-(tert-butoxycarbonylamino)propionyl]amino-4-flurobicyclo[3.1.0]hexane-2,6-dicarboxylicacid ethyl ester (88.16 g, 191 mmol) in 238 mL of tetrahydrofuran atroom temperature is added 238 mL (477 mmol) of 2N sodium hydroxide. Thebiphasic mixture is allowed to stir vigorously at room temperature for2.5 hours at which time the reaction is homogeneous. The mixture isdiluted with 238 mL of 1-butyl methyl ether followed by mixing andseparation of the layers. The aqueous layer is further diluted with 238mL of water and filtered to remove particulate matter. The solution istreated with concentrated HCl (42.9 mL, 515 mmol) over 30 minutesoptionally followed by seeding with the title compound and stirring for1 hour. The resultant slurry is filtered, washed with water (2×100 mL),and vacuum dried at 45° C. for 40 hours to provide 72.2 g of the titlecompound as a white solid. A portion of the solid (69.5 g) is allowed tostir with 490 mL of acetone for 1 hour to produce a hazy solution thatis filtered, washing with acetone (2×100 mL). The filtrate isconcentrated in vacuo to a white foam which is further dried in vauo at45° C. for 16 hours to provide 61.8 g (corrected for 12% wt/wt acetone)of the title compound.

EXAMPLE 1 (1R,4S,5S,6S)-4-(2′S-Aminopropionyl)amino-]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

To a suspension of(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (110.0 g, 271 mmol, Preparation 3) in 563 mL of ethyl acetate addeda solution of hydrogen chloride in ethyl acetate (3.7 M, 514 mL) over 20minutes. After the suspension stirs for 2.5 h, filter and wash the cakewith ethyl acetate (1×200 mL, 1×115 mL). After vacuum drying at 46° C.for 18 hours, the title compound is collected as a white solid (85.77 g,92%).

¹H NMR (300 MHz, Methanol-d₄) δ: 4.12 (brd, 1H, J=14.6 Hz), 3.94 (q, 1H,J=7.1 Hz), 3.52 (ddd, 1H, J=7.0, 3.9, 0.9 Hz), 3.16 (d, 1H, J=14.6 Hz),3.02 (dd, 1H, J=7.0, 4.4 Hz), 2.49 (t, 1H, J=4.1 Hz), 1.52 (d, 3H, J=7.1Hz).

EXAMPLE 2(1R,4S,5S,6S)-4-(2′S-2′-Aminopropionyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid tosylate

A suspension of(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (300 mg, 0.738 mmol, Preparation 3) and toluenesulfonic acidmonohydrate (140 mg, 0.738 mmol) in toluene (3 mL) is heated to 75° C.and stirred for 45 min before it is allowed to cool to room temperatureand stir for 16 hours. Filter the suspension and wash the cake withtoluene (2×1 mL). After vacuum drying at 45° C. for 1 hour, 307 mg (87%)of the title compound is collected as a white solid.

mp (DSC) 233° C.

¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, 2H, J=8.5 Hz), 7.24 (d, 2H, J=8.0Hz), 4.11 (d, 1H, J=15 Hz), 3.94 (q, 1H, J=7.0 Hz), 3.53 (dd, 1H, J=7.0,4.0 Hz), 3.13 (dd, 1H, J=14, 1.0 Hz), 3.02 (dd, 1H, J=7.0, 4.5 Hz), 2.48(t, 1H, J=4.5 Hz), 2.37 (s, 3H), 1.52 (d, 3H, J=7.5 Hz).

¹³C NMR (125 MHz, CD₃OD) δ 170.70, 170.32, 169.80, 142.04, 140.78,128.79, 125.79, 60.20, 54.73, 48.77, 42.44, 30.84, 22.22, 20.20, 16.09.

EXAMPLE 3(1R,4S,5S,6S)-4-(2′S-Amino-3′-hydroxy-propionyl)-amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloric acid

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(3′-acetoxy-2′S-tert-butoxycarbonylamino-propionyl)-amino-2,2-dioxo-2λ⁶-thiabicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (380 mg, 0.77 mmoles, Preparation 15).

[α]_(D) ²³=−19.23 (c=0.52, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 2.48 (1H, t, J=4.0 Hz), 3.04 (1H, dd, J=4.4,7.3 Hz), 3.17 (1H, d, J=14.7 Hz), 3.51 (1H, dd, J=4.4, 7.0 Hz), 3.77(1H, dd, J=7.0, 10.6 Hz), 3.94-4.12 (3H, m).

Anal Calcd for C₁₀H₁₄N₂O₈S.HCl.H₂O: C, 31.88; H, 4.55; N, 7.44; Cl,9.41. Found: C, 31.53; H, 4.40; N, 7.32; Cl, 9.24.

HRMS calcd for C₁₀H₁₅N₂O₈S, 323.0549. Found, 323.0533.

EXAMPLE 4(1R,4S,5S,6S)-4-[(Pyrrolidine-2′S-carbonyl)-amino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C employing(1R,4S,5S,6S)-4-[(1′-tert-butoxycarbonyl-pyrrolidine-2′S-carbonyl)-amino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid dimethyl ester (0.8 g, 1.7 mmol, Preparation 11) to yield 0.42 g(67.0%) of the title compound.

[α]_(D) ²³=−32 (c=1.00, CH₃OH).

¹H NMR (300 MHz, CD₃OD) δ 1.98-2.12 (3H, m), 2.40-2.50 (1H, m), 2.52(1H, t, J=4.4 Hz), 2.99 (1H, dd, J=4.4, 7.0 Hz), 3.2 (1H, d, J=14.7 Hz),3.29-3.42 (3H, m), 3.54 (1H, m), 4.13 (1H, d, J=15.8 Hz), 4.30 (1H, dd,J=6.2, 9.2 Hz).

HRMS calcd for C₁₂H₁₇N₂O₇S, 333.0756. Found, 333.0740.

EXAMPLE 5(1R,4S,5S,6S)-4-(2′S-Amino-4′-methylsulfanyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C employing(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-4′-methylsulfanyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.77 g, 1.6 mmol, Preparation 12) to yield 0.41 g(54.9%) of the title compound.

[α]_(D) ²³=+4 (c=1.00, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 2.1-2.2 (2H, m), 2.13 (3H, s), 2.47 (1H, t,J=4.4 Hz), 2.58-2.63 (2H, m), 3.02 (1H, dd, J=4.0, 7.0 Hz), 3.12 (1H, d,J=14.7 Hz), 3.52 (1H, dd, J=3.3, 6.6 Hz), 3.98 (1H, t, J=6.2 Hz), 4.14(1H, d, J=14.7 Hz).

HRMS calcd for C₁₂H₁₉N₂O₇S₂, 367.0634. Found, 367.0634.

EXAMPLE 6(1R,4S,5S,6S)-4-[2′S-Amino-3′-(1H-indol-3-yl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C employing(1R,4S,5S,6S)-4-[2′S-tert-butoxycarbonylamino-3′-(1-tert-butoxycarbonyl-1H-indol-3-yl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid dimethyl ester (0.54 g, 0.83 mmol, Preparation 13) to yield 0.28 g(73.6%) of the title compound.

[α]_(D) ²³=+7.8 (c=1.02, CH₃OH).

¹H NMR (300 MHz, CD₃OD) δ 2.47-2.53 (1H, m), 3.05-3.18 (3H, m),3.44-3.57 (2H, m), 4.13-4.23 (2H, m), 7.07-7.41 (3H, m), 7.71-7.78 (2H,m), 8.22 (1H, app d, J=7.7 Hz).

Anal Calcd for C₁₈H₉N₃O₇S.HCl: C, 47.22; H, 4.40; N, 9.18. Found: C,46.51; H, 3.96; N, 8.54.

MS (ES) m/z 420.1 [M−1]⁻.

EXAMPLE 7(1R,4S,5S,6S)-4-[2′S-Amino-3′-(4-hydroxy-phenyl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C employing(1R,4S,5S,6S)-4-[2′S-tert-butoxycarbonylamino-3′-(4-tert-butoxycarbonyloxy-phenyl)-propionylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane-4,6-dicarboxylicacid dimethyl ester (0.33 g, 0.53 mmol, Preparation 14) to yield 0.13 g(56.4%) of the title compound.

[α]_(D) ²³=−6 (c=1.00, H₂O).

¹H NMR (300 MHz, CD₃OD) δ 2.47 (1H, t, J=4.0 Hz), 2.87 (1H, dd, J=9.2,14.7 Hz), 3.05-3.12 (2H, m), 3.23 (1H, dd, J=5.1, 14.7 Hz), 3.55 (1H,dd, J=4.0, 7.0 Hz), 4.00 (1H, dd, J=4.8, 9.2 Hz), 4.13 (1H, d, J=14.7Hz), 6.80 (2H, d, J=8.4 Hz), 7.13 (2H, d, 8.8 Hz).

HRMS calcd for C₁₆H₁₉N₂O₈S, 399.0862. Found, 399.0844.

EXAMPLE 8(1R,4S,5S,6S)-4-(2′S-Amino-3′-phenyl-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-3′-phenyl-propionylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 5). Yield 0.55 g (85%, 1.31 mmol) of awhite solid.

[α]_(D) ²³=+4.17 (c=0.48, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 2.45 (1H, t, 1=4.0 Hz), 2.98 (1H, dd, J=9.2,14.7 Hz), 3.06 (1H, dd, J=4.4, 7.0 Hz), 3.10 (1H, d, J=14.6 Hz), 3.33(1H, dd, J=4.8, 13.6 Hz), 3.52 (1H, dd, J=3.3, 7.3 Hz), 4.08 (1H, dd,J=5.1, 8.8 Hz), 4.10 (1H, d, J=15.4 Hz), 7.31-7.42 (5H, m).

¹³C NMR (300 MHz, D₂O w/1,4-dioxane): δ 172.55, 171.94, 170.01, 134.31,130.39, 130.27, 130.01, 129.00, 61.38, 54.76, 54.37, 43.07, 37.36,31.57, 23.18.

Anal Calcd for C₁₆H₁₈N₂O₇S.1.5HCl: C, 43.97; H, 4.50; N, 6.41. Found: C,43.59; H, 4.17; N, 6.46.

MS (ES) m/z found 383.1 [M+H]⁺.

HRMS calcd for C₁₆H₁₉N₂O₇S [M+H]⁺: 383.0913. Found: 383.0923.

EXAMPLE 9(1R,4S,5S,6S)-4-(2′S-Amino-3′S-methyl-pentanonylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-3′S-methyl-pentanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 6). Yield 0.43 g (80%, 1.12 mmol) of awhite solid.

[α]_(D) ²³=+4.08 (c=0.49, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 0.98 (3H, t, J=7.3 Hz), 1.07 (3H, d, J=7.0Hz), 1.15-1.24 (1H, m), 1.53-1.62 (1H, m), 1.95-2.04 (1H, m), 2.44 (1H,t, J=4.0 Hz), 3.06 (1H, dd, J=4.4, 7.0 Hz), 3.14 (1H, d, J=15.0 Hz),3.52 (1H, dd, J=3.7, 7.0 Hz), 3.73 (1H, d, J=5.1 Hz), 4.10 (1H, d,J=14.7 Hz).

Anal Calcd for C₁₃H₂₀N₂O₇S.HCl.0.5H₂O: C, 39.64; H, 5.63; N, 7.11.Found: C, 39.38; H, 5.39; N, 7.04.

MS (ES) m/z found 349.1 [M+H]⁺.

HRMS (ES) calcd for C₁₃H₂₁N₂O₇S [M+H]⁺, 349.1069. Found, 349.1086.

EXAMPLE 10(1R,4S,5S,6S)-4-(2′S-Amino-3′-methyl-butylamino)-2,2-dioxo-2)₆-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-3′-methyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 7). Yield 0.18 g (88%, 0.49 mmol) of awhite solid as the HCl salt.

[α]_(D) ²³=+7.84 (c=0.51, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 1.04 (3H, d, J=6.6 Hz), 1.09 (3H, d, J=7.0Hz), 2.22-2.29 (1H, m), 2.44 (1H, t, J=4.0 Hz), 3.05 (1H, dd, J=4.4, 7.0Hz), 3.10 (1H, d, J=14.7 Hz), 3.52 (1H, dd, J=4.0, 7.3 Hz), 3.67 (1H, d,J=5.5 Hz), 4.10 (1H, d, J=14.7 Hz).

Anal Calcd for C₁₂H₁₈N₂O₇S.HCl.0.4H₂O: C, 38.12; H, 5.20; N, 7.41.Found: C, 37.78; H, 4.90; N, 7.15.

MS (ES) m/z found 335.1 [M+H]⁺.

EXAMPLE 11(1R,4S,5S,6S)-4-(2′S-Amino-4′-methyl-pentanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(2′S-tert-butoxycarbonylamino-4′-methyl-pentanoylamino)-2,2-dioxo-2,6-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 8). Yield 0.50 g (76%, 1.30 mmol) of awhite solid as the HCl salt.

[α]_(D) ²³=−4.0 (c=0.50, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 1.00 (3H, d, J=5.5 Hz), 1.02 (3H, d, J=5.9Hz), 1.62-1.79 (3H, m), 2.42 (1H, t, J=4.0 Hz), 3.04 (1H, dd, J=4.4, 7.3Hz), 3.13 (1H, d, J=15.0 Hz), 3.52 (1H, dd, J=3.3, 7.0 Hz), 3.84-3.89(1H, m), 4.10 (1H, d, J=15.0 Hz).

Anal Calcd for C₁₃H₂₀N₂O₇S.HCl.0.3H₂O: C, 40.01; H, 5.58; N, 7.18.Found: C, 39.66; H, 5.57; N, 6.99.

MS (ES) m/z found 349.1 [M+H]⁺.

(1R,4S,5S,6S)-4-(2′S,6′-Diamino-hexanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid bis hydrochloride

Prepare according to General Procedure C using(1R,4S,5S,6S)-4-(2′S,6′-bis-tert-butoxycarbonylamino-hexanoylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (Preparation 9). Yield 0.56 g (86%, 1.28 mmol) of awhite solid as the bis hydrochloride salt.

[α]_(D) ²³=−4.0 (c=0.50, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 1.48-1.55 (2H, m), 1.67-1.74 (2H, m),1.89-1.97 (2H, m), 2.47 (1H, t, J=4.0 Hz), 2.97 (2H, app t, J=4.0), 3.08(1H, dd, J=4.4, 7.0 Hz), 3.20 (1H, d, J=15.0 Hz), 3.53 (1H, dd, J=3.7,7.0 Hz), 3.93 (1H, app. t, J=6.2 Hz), 4.08 (1H, d, J=14.7 Hz).

Anal Calcd for C₁₃H₂₁N₃O₇S.2HCl.0.2H₂O: C, 35.49; H, 5.36; N, 9.55.Found: C, 35.30; H, 5.48; N, 9.42.

MS (ES) m/z found 364.1 [M+H]⁺.

EXAMPLE 13(1R,4S,5S,6S)-4-(2′S-Amino-4′-carbamoyl-butyrylamino)-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride

Stir(1R,4S,5S,6S)-4-[2′S-tert-butoxycarbonylamino-4′-(trityl-carbamoyl)-butyrylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid dimethyl ester (0.48 g, 0.65 mmol, Preparation 10) in a 1:1 mixtureof 2.5 N LiOH and THF (6 mL total volume) at room temperature for 4hours. Adjust the reaction mixture to pH=2 with 1N HCl and extract theproduct with ethyl acetate. Combine all organics, wash with brine, dryover MgSO₄ and concentrate to yield 0.46 g of(1R,4S,5S,6S)-4-[2′S-tert-butoxycarbonylamino-4′-(trityl-carbamoyl)-butyrylamino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid as a white foam. Dissolve the diacid in DCM and stir at roomtemperature as anisole (0.28 g, 2.6 mmol) and then TFA (3.70 g, 32.5mmol) are consecutively added. Stir the resulting reaction mixture atroom temperature for 2 hours and concentrated in vacuo. Triturate theresulting yellow oil in Et₂O until a freely flowing white precipitateforms. Collect the TFA salt by vacuum filtration under N₂ blanket.Dissolve product in 1 mL 1N HCl and subject to lyophilization to affordthe desired product as the HCl salt. Yield 0.16 g (62%, 0.40 mmol) of awhite solid.

[α]_(D) ²³=+8.0 (c=1.0, H₂O).

¹H NMR (300 MHz, CD₃OD) δ 2.00-2.08 (2H, m), 2.37-2.41 (3H, m), 2.93(1H, dd, J=4.4, 7.3 Hz), 3.04 (1H, d, J=14.7 Hz), 3.45 (1H, dd, J=3.7,7.0 Hz), 3.86 (1H, app. t, J=5.9 Hz), 4.05 (1H, d, J=14.7 Hz).

Anal Calcd for C₁₂H₁₇N₃O₈S.HCl.2.0H₂O: C, 33.07; H, 5.09; N, 9.64.Found: C, 33.37; H, 4.69; N, 9.39.

MS (ES) m/z found 363.9 [M+H]⁺.

HRMS (ES) calcd for C₁₂H₁₈N₃O₈S [M+H]⁺: 364.0815. Found: 364.0825.

EXAMPLE 14(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Stir a slurry of(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (53.0 g corrected for acetone, 142 mmol) in 447 mL of acetone at50° C. for 35 minutes. Filter the hazy solution to clarify the solution,followed by rinsing with 100 mL of acetone. Add 22.1 mL (265 mmol) ofconcentrated hydrochloric acid dropwise to the clear, off-white filtrateover 5 minutes. Warm the mixture to 45-50° C. and stir for 90 minutes.Optionally seed the mixture with(1R,2S,4R,5R,6R)-2-(2′S-aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride, followed by turning off the heat and allowing togradually cool to room temperature. After 2 hours the temperaturereaches 25° C. and acetone (942 mL) is added to the slurry over 90minutes. Stir the slurry an additional 16 hours, followed by filtration,washing with acetone (2×200 mL), and vacuum drying at 45° C. for 9 hoursand at room temperature for another 64 hours to produce 40.2 g (91%) ofthe title compound as a white solid.

Recrystallization

Dissolve 1.06 g in 0.5 mL of water and 2.12 mL of acetone with heatingat 50° C. followed by dilution with another 5.3 mL of acetone andoptionally seeding. Add another 4.2 mL of acetone to the faintly cloudymixture optionally followed again by seeding, turning off the beat, andallowing gradual cooling to room temperature over 1 hour. Dilute theresultant slurry further with another 9.5 mL of acetone over 30 minutesfollowed by stirring for 15 hours. Upon filtering, washing with acetone(2×5 mL), and vacuum drying at 45° C. for 10 hours and at roomtemperature for 60 hours, 0.905 g (85% recovery) of the title compoundis obtained as a white solid.

mp (DSC) 183° C.

[α]²⁵ _(D) +33° (c 1.06, CH₃OH).

500 MHz ¹H NMR (CD₃OD) δ 5.58-5.42 (m, 1H), 3.92 (q, 1H, J=7.0 Hz), 2.96(dd, 1H, J=14, 8.0 Hz), 2.41-2.39 (m, 1H), 2.35-2.30 (m, 1H), 2.10 (t,1H, J=3.0 Hz), 1.52 (d, 3H, J=7.5 Hz), 1.51-1.42 (m, 1H).

¹³C NMR (125 MHz, CD₃OD) δ 173.74, 173.62, 170.00, 93.48 and 92.04 (C—Fsplitting), 63.95 and 63.92 (C—F splitting), 48.80, 36.89 and 36.70 (C—Fsplitting), 32.97 and 32.91 (C—F splitting), 30.05 and 29.87 (C—Fsplitting), 19.37, 16.28.

FTIR (DRIFT) 3430 (w), 3016 (s), 1721 (s), 1662 (s), 1496 (s), 1190 (m),1024 (m), 637 (w) cm⁻¹.

EXAMPLE 15(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate

A slurry of(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (1.87 g corrected, 4.98 mmol, Preparation 18) in 16.8 mL of acetoneis allowed to stir at 50° C. for 15 minutes. The hazy solution isfiltered to clarify the solution followed by rinsing with acetone(3×1.25 mL). The clear filtrate is diluted with 0.935 mL of water,placed in a heating bath at 50° C., and treated with 0.647 mL (9.97mmol) of methanesulfonic acid dropwise (gas evolution observed). A whiteslurry is produced after 25 minutes. After stirring a total of 2 hours,another 35.5 mL of acetone is added over 5-10 minutes. The heat isturned off and the slurry is allowed to cool gradually to roomtemperature over 2 hours followed by filtration, washing with acetone(2×8 mL), and vacuum drying at 45° C. for 14 hours to give 1.77 g (95%)of the title compound as a faint pink solid. A sample of this materialis recrystallized as follows: 1.65 g is dissolved in 1.16 mL of waterand 4.95 mL of acetone with heating at 50° C. followed by dilution withanother 1.65 mL of acetone and optionally seeding. The heat is turnedoff and the mixture is allowed to gradually cool to room temperature.Acetone (26.4 mL) is added simultaneously over 40 min. The resultantslurry is allowed to stir an additional 3 hours. Upon filtering, washingwith acetone (2×6 mL), and vacuum drying at 45° C. for 6 h and at roomtemperature for 60 hours, 1.59 g (96% recovery) of the title compound isobtained as a white solid:

mp (DSC) 206° C.

[α]²⁵ _(D) +3° (c 1.05, CH₃OH).

¹H NMR (500 MHz, CD₃OD) δ 5.58-5.42 (m, 1H), 3.92 (q, 1H, J=7.0 Hz),2.96 (dd, 1H, J=14, 8.0 Hz), 2.70 (s, 3H), 2.41-2.39 (m, 1H), 2.35-2.30(m, 1H), 2.10 (t, 1H, J=3.0 Hz), 1.52 (d, 3H, J=7.5 Hz), 1.51-1.42 (m,1H).

¹³C NMR (125 MHz, CD₃OD) δ 173.73, 173.61, 170.02, 93.50 and 92.05 (C—Fsplitting), 63.91, 48.79, 38.30, 36.89 and 36.70 (C—F splitting), 32.97and 32.91 (C—F splitting), 30.02 and 29.84 (C—F splitting), 19.37,16.26.

FTIR (DRIFT) 3472 (w), 3077 (s), 1717 (s), 1691 (s), 1557 (m), 1220 (s),1019 (m), 781 (m), 563 (m) cm⁻¹.

Anal. Cald for C₁₂H₁₉FN₂O₈S: C, 38.92; H, 5.17; N, 7.56. Found: C,38.96; H, 4.97; N, 7.51.

EXAMPLE 16(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid esylate

A slurry of(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (0.2 g, 0.534 mmol, Preparation 18) in 1.8 mL of acetone is allowedto stir at 50° C. for 5 minutes. The hazy solution is filtered toclarify the solution followed by rinsing with acetone (1×0.4 mL). Theclear filtrate is diluted with 0.1 mL of water, placed in a heating bathat 50° C., and treated with 0.124 mL (1.07 mmol) of ethanesulfonic aciddropwise (gas evolution observed). A white slurry is produced after 90minutes. After stirring a total of 2 hours, another 1.8 mL of acetone isadded over 5 min. The heat is turned off and the slurry is allowed tocool gradually to room temperature over 1 hours followed by stirring anadditional 2 hours. Filtration, washing with acetone (2×1 mL), andvacuum drying at 45° C. for 4 hours and at room temperature for 60 hoursafforded 0.173 g (84%) of the title compound as a white solid.

mp (DSC) 210° C. (decomp).

¹H NMR (500 MHz, CD₃OD) δ 5.58-5.42 (m, 1H), 3.92 (q, 1H, J=7.0 Hz),2.96 (dd, 1H, J=14, 8.0 Hz), 2.80 (q, 2H, 7.3 Hz), 2.42-2.37 (m, 1H),2.35-2.30 (m, 1H), 2.09 (t, 1H, J=3.0 Hz), 1.52 (d, 3H, J=7.5 Hz),1.51-1.40 (m, 1H), 1.30 (t, 3H, J=7.5 Hz).

EXAMPLE 17(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid besylate

A slurry of(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (0.402 g 1.07 mmol, Preparation 18) in 3.6 mL of acetone is allowedto stir at 50° C. for 10 minutes. The hazy solution is treated with asmall scoop of celite and filtered to clarify the solution followed byrinsing with acetone (2×0.4 mL). The clear filtrate is placed in aheating bath at 50° C., and treated with 226 mg (90%, 1.29 mmol) ofbenzenesulfonic acid as a solution in 0.113 mL of water followed by arinse with 0.4 mL of acetone (gas evolution observed). After stirring atgentle reflux for 4 hours, the heat is turned off and the reaction istreated with 8 mL of acetone over 10 minutes optionally followed byseeding. A slurry had formed over 1 hour which is diluted with 3.2 mL ofacetone followed by stirring at room temperature an additional 15.5hours. Filtration, washing with acetone (2×10 mL) and drying in vacuo at45° C. for 24 h provided 313 mg (62% corrected for 10 wt % acetone) ofthe title compound as a white solid.

mp (DSC) 132° C.

¹H NMR (500 MHz, CD₃OD) δ 7.86-7.80 (m, 2H), 7.46-7.37 (m, 3H),5.58-5.42 (m, 1H), 3.92 (q, 1H, J=7.0 Hz), 2.96 (dd, 1H, J=14, 8.0 Hz),2.42-2.37 (m, 1H), 2.35-2.30 (m, 2H), 2.09 (t, 1H, J=3.0 Hz), 1.52 (d,3H, J=7.5 Hz), 1.51-1.40 (m, 1H).

EXAMPLE 18(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid tosylate

A slurry of(1R,2S,4R,5R,6R)-2-[2′S-(tert-butoxycarbonylamino)propionyl]-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid (1.04 g corrected, 2.78 mmol, Preparation 18) in 9.36 mL of acetoneis allowed to stir at 50° C. for 15 minutes. The hazy solution istreated with a small scoop of celite and filtered to clarify thesolution followed by rinsing with acetone (1×2.08 mL then 1×1.04 mL).The clear filtrate is placed in a heating bath at 50° C., and treatedwith 634 mg (3.33 mmol) of p-toluenesulfonic acid monohydrate as asolution in 0.317 mL of water followed by a rinse with 0.317 mL ofacetone (gas evolution observed). After stirring at gentle reflux for 4hours, the reaction is removed from the heating bath and treated with10.4 mL of acetone over 10 minutes. The clear, colorless solution isoptionally seeded and a precipitate is observed to form over 30 min atwhich time another 10.4 mL of acetone is introduced over 20 minutes. Theslurry is allowed to stir an additional 4 hours followed by filtration,washing with acetone (2×10 mL) and drying in vacuo at 45° C. for 14hours to provide 995 mg (78% corrected for 3 wt % acetone) of the titlecompound as a white solid.

mp (DSC) 155° C.

¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, 2H, J=7.5 Hz), 7.34 (d, 2H, J=8.5Hz), 5.58-5.42 (m, 1H), 3.92 (q, 1H, J=7.0 Hz), 2.96 (dd, 1H, J=14, 8.0Hz), 2.42-2.30 (m, 2H), 2.24 (s, 3H), 2.09 (t, 1H, J=3.0 Hz), 1.52 (d,3H, J=7.5 Hz), 1.51-1.40 (m, 1H).

EXAMPLE 19(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid

To solution of(1R,2S,4R,5R,6R)-2-(2′S-amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate (0.5 g, 1.35 mmol, Example 15) in 1 mL of water at 50° C.is added 5 mL of 3 A ethanol followed after a few minutes by 0.27 mL(1.35 mmol) of 5 N aqueous sodium hydroxide. The beat is turned off andthe clear colorless solution is diluted with 2.5 mL of ethanol,optionally seeded, and diluted further with 7.5 mL of ethanol over 30min. The resulting slurry is allowed to stir thereafter with cooling toroom temperature over 1 h and subsequently at room temperature for 2hours. The solid is collected and washed with ethanol (1×10 mL) followedby drying in vacuo at 45° C. for 18.5 hours to afford 0.301 g (78% yieldcorrected for 1.6 wt % sodium methanesulfonate and 3 wt % ethanol) ofthe title compound as a white solid.

¹H NMR (500 MHz, D₂O) δ 5.45-5.30 (m, 1H), 3.88 (q, 1H, J=7.0 Hz), 2.58(dd, 1H, J=14, 8.0 Hz), 2.33-2.30 (m, 1H), 2.27-2.26 (m, 1H), 1.92 (t,1H, J=3.0 Hz), 1.36 (d, 3H, J=7.1 Hz), 1.41-1.32 (m, 1H).

¹³C NMR (125 MHz, D₂O) δ 177.46, 176.92, 170.42, 94.56 and 93.19 (C—Fsplitting), 65.36, 49.01, 36.75 and 36.57 (C—F splitting), 33.61 and33.55 (C—F splitting), 30.54 and 30.36 (C—F splitting), 20.27, 16.67.

EXAMPLE 20(1R,2S,4R,5R,6R)-2-(2′S-Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicdisodium salt

To a solution of(1R,2S,4R,5R,6R)-2-(2′S-amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate (70 mg, 0.19 mmol, Example 15) in 420 μL of methanol at60° C. is added a warm solution of sodium acetate (46.5 mg, 0.57 mmol)in 470 μL of methanol with a rinse of 230 μL of methanol. The heat isturned off. The stirring hazy solution is diluted with 280 μL ofmethanol optionally followed by seeding to aid the crystallization. Theresulting slurry is slowly cooled to ambient temperature over 1 hour andstirred for 2 hours at ambient temperature. The product is isolated byfiltration, washed with methanol (2×280 μL), and dried in vacuo at 45°C. for 15 hours to furnish 52.5 mg (85% yield corrected for 2.3 wt %sodium methanesulfonate and 0.2 wt % methanol) of the title compound asa white solid.

¹H NMR (500 MHz, D₂O) 5.44-5.29 (m, 1H), 3.89 (q, 1H, J=7.0 Hz), 2.65(s, 3H), 2.56 (dd, 1H, J=14, 8.0 Hz), 2.16-2.13 (m, 1H), 2.10-2.09 (m,1H), 1.74 (t, 1H, J=3.1 Hz), 1.38 (d, 3H, J=7.1 Hz), 1.36-1.28 (m, 1H).

¹³C NMR (125 MHz, D₂O) 180.00, 178.72, 170.13, 95.40 and 93.99 (C—Fsplitting), 65.97, 49.06, 37.25 and 37.07 (C—F splitting), 33.01 and32.94 (C—F splitting), 29.64 and 29.46 (C—F splitting), 22.48, 16.68.

EXAMPLE 21(1S,2S,4S,5R,6R)-2-(2′S-aminopropionyl)amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

To a solution of(1S,2S,4S,5R,6R)-4-acetyloxy-2-[2′S-(tert-butoxy)carbonylaminopropionyl]-aminobicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (0.600 g, 1.28 mmol, Preparation 21) in THF (30 mL)and water (30 mL) is added lithium hydroxide monohydrate (0.535 g, 12.8mmol). After stirring at room temperature for 3 hours, the reaction isdiluted with water and washed with ethyl acetate (2×25 mL). The aqueouslayer is acidified to pH 1 with aqueous 1N HCl solution and extractedwith ethyl acetate (3×30 mL). The combined organic extracts areconcentrated, redissolved in 1N HCl in ethyl acetate (60 mL), thenstirred under nitrogen at room temperature for 16 hours. The solvent isremoved in vacuo to afford 400 mg (99%) of the product as a white solid.

LCMS: m/z 273 [M+H]⁺ @ R_(T) 0.20 min.

¹H NMR (CD₃OD)*: 4.13 (1H, d, 5.9 Hz), 3.80 (1H, q, 6.7 Hz), 2.41-2.31(2H, m), 1.93 (1H, dd, 6.0 Hz, 2.7 Hz), 1.62 (1H, dd, 5.9 Hz, 15.5 Hz),1.44 (1H, t, 3.0 Hz), 1.38 (3H, d, 6.8 Hz); *N.B. Exchangeable protonsnot observed by NMR=6.

EXAMPLE 22(1S,2S,4S,5R,6R)-2-(2′-Amino-acetylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′-tert-butoxycarbonylamino-acetylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (300 mg, 0.84 mmoles, Preparation 23), with theexception that the tert-butoxycarbonyl protecting group is removed bytreating with 4M HCl in dioxane. Yield 156 mg (63%).

[α]_(D) ²³=−36 (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.56 (1H, t, J=2.9 Hz), 1.74 (1H, dd, J=5.8,15.4 Hz), 2.12 (1H, m), 2.48 (1H, d, J=15.4 Hz), 2.61 (1H, m), 3.62 (2H,s), 4.32 (1H, d, J=5.49 Hz).

Anal Calcd for C₁₀H₁₄N₂O₆.1.3HCl.H₂O: C, 37.11; H, 5.39; N, 8.66. Found:C, 37.36; H, 4.99; N, 8.30.

MS (ES) m/z 258.8 [M+H]⁺.

EXAMPLE 23(1S,2S,4S,5R,6R)-2-(2′S-Amino-3′-methyl-butyrylamino)-4-hydroxybicyclo[3.1.0]-hexane-2,6-dicarboxylicacid hydrochloric acid

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S-tert-butoxycarbonylamino-3′-methyl-butyrylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (480 mg, 1.19 mmoles, Preparation 24), with theexception that the tert-butoxycarbonyl protecting group is removed bytreating with 4M HCl in dioxane. Yield 307 mg (76%).

[α]_(D) ²³=+8.33 (c=0.48, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.07 (3H, d, J=6.8 Hz), 1.11 (3H, d, J=7.3Hz), 1.55 (1H, t, J=2.9 Hz), 1.76 (1H, dd, J=5.8, 15.6 Hz), 2.14 (1H,dd, J=3.4, 5.8 Hz), 2.24 (1H, m), 2.50 (1H, d, J=15.6 Hz), 2.64 (1H, dd,J=2.9, 5.8 Hz), 3.66 (1H, d, J=5.4 Hz), 4.32 (1H, d, J=5.8 Hz).

Anal Calcd for C₁₃H₂₀N₂O₆.HCl.1.1H₂O: C, 43.79; H, 6.56; N, 7.86. Found:C, 43.77; H, 6.20; N, 7.47.

HRMS calcd for C₁₃H₂₁N₂O₆, 301.1400. Found, 301.1400.

EXAMPLE 24(1S,2S,4S,5R,6R)-2-(2′S-Amino-4′-methyl-pentanoylamino)₄-hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylicacid hydrochloric acid

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S-tert-butoxycarbonylamino-4′-methyl-pentanoylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (460 mg, 1.11 mmoles, Preparation 25), with theexception that the tert-butoxycarbonyl protecting group is removed bytreating with 4M HCl in dioxane. Yield 371 mg (95%).

[α]_(D) ²³=+4 (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.01 (3H, d, J=5.8 Hz), 1.03 (3H, d, J=6.3Hz), 1.54 (1H, t, J=2.9 Hz), 1.63-1.82 (4H, m), 2.14 (1H, dd, J=2.9, 5.8Hz), 2.49 (1H, d, J=15.6 Hz), 2.62 (1H, dd, J=2.9, 5.8 Hz), 3.83-3.86(1H, m), 4.32 (1H, d, J=5.8 Hz).

Anal Calcd for C₁₄H₂₂N₂O₆.HCl.1.4H₂O: C, 44.72; H, 6.92; N, 7.45. Found:C, 44.52; H, 6.57; N, 7.13.

HRMS calcd for C₁₄H₂₃N₂O₆, 315.1556. Found, 315.1569.

EXAMPLE 25(1S,2S,4S,5R,6R)-2-(2′S-Amino-3′S-methyl-pentanoylamino)-4-hydroxy-bicyclo[3.1.0]-hexane-2,6-dicarboxylicacid hydrochloric acid

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S-tert-butoxycarbonylamino-3′S-methyl-pentanoylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (410 mg, 0.99 mmoles, Preparation 26), with theexception that the tert-butoxycarbonyl protecting group is removed bytreating with 4M HCl in dioxane. Yield 330 mg (95%).

[α]_(D) ²³=+8 (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 0.99 (3H, t, J=7.3 Hz), 1.08 (3H, t, J=7.3Hz), 1.17-1.27 (1H, m), 1.53 (1H, t, J=2.9 Hz), 1.59-1.65 (1H, m), 1.76(1H, dd, J=5.8, 15.6 Hz), 1.96-2.00 (1H, m), 2.14 (1H, dd, J=2.9, 5.8Hz), 2.46 (1H, d, J=15.6 Hz), 2.66 (1H, dd, J=2.9, 5.8 Hz), 3.70 (1H, d,J=5.4 Hz), 4.31 (1H, d, J=5.8 Hz).

Anal Calcd for C₁₄H₂₂N₂O₆1.1HCl.1.2H₂O: C, 44.71; H, 6.83; N, 7.45.Found: C, 44.38; H, 6.51; N, 7.08.

HRMS calcd for C₁₄H₂₃N₂O₆, 315.1556. Found, 315.1566.

EXAMPLE 26(1S,2S,4S,5R,6R)-2-[2′-(2-Amino-acetylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Treat(1S,2S,4S,5R,6R)-2-[2′-(2-tert-butoxycarbonylamino-acetylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (360 mg, 0.77 mmoles, Preparation 27) with an excessof 4M HCl in dioxane and stir for 40 minutes; concentrated in vacuo. Addethyl acetate and concentrated. Treat the solid with THF (3M) and 1NLiOH (3.0 equiv.). Stir for 1 hour then add 1N HCl until pH=3.Concentrated the reaction in vacuo. Purify via DOWEX® 50WX8-100ion-exchange resin. Yield 188 mg (78%).

[α]_(D) ²³=+3.92 (c=0.51, H₂O).

¹H NMR 300 MHz, D₂O) δ 1.43 (1H, t, J=2.9 Hz), 1.53 (1H, dd, J=5.9, 15.4Hz), 1.98 (1H, m), 2.23-2.31 (2H, m), 3.70 (2H, s), 3.82 (2H, app d, J=1Hz), 4.16 (1H, d, J=5.9 Hz).

HRMS calcd for C₁₂H₁₈N₃O₇, 316.1145. Found, 316.1123.

EXAMPLE 27(1S,2S,4S,5R,6R)-2-[2′-(2S-Amino-propionylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Prepare according to the general procedure outlined in Example 26 using(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-butoxycarbonylamino-propionylamino)-acetylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (410 mg, 0.84 mmoles, Preparation 28). Yield 200 mg(72%).

[α]_(D) ²³=+23.53 (c=0.51, H₂O).

¹H NMR, 300 MHz, D₂O) δ 1.36 (3H, d, J=7.0 Hz), 1.37 (1H, t, J=3.3 Hz),1.49 (1H, dd, J=5.9, 15.4 Hz), 1.91 (1H, dd, J=2.9, 5.9 Hz), 2.15 (1H,dd, J=2.9, 5.9 Hz), 2.27 (1H, d, J=15.4 Hz), 3.79 (2H, s), 3.95 (1H, q,J=7.0 Hz), 4.13 (1H, d, J=5.5 Hz).

HRMS calcd for C₁₃H₁₉N₃O₇, 330.1301. Found, 330.1290.

EXAMPLE 28(1S,2S,4S,5R,6R)-2-(2′S-Amino-3′-phenyl-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′-tert-butoxycarbonylamino-3′-phenyl-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (400 mg, 0.89 mmoles, Preparation 29). Yield 290 mg(85%).

[α]_(D) ²³=+3.64 (c=0.55, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.45 (1H, t, J=3.3 Hz), 1.60 (1H, dd, J=5.5,15.4 Hz), 2.03 (1H, dd, J=2.6, 5.9 Hz), 2.37 (1H, d, J=15.4 Hz), 2.55(1H, dd, J=2.9, 5.9 Hz), 2.90 (1H, dd, J=8.4, 14.3 Hz), 3.22 (1H, dd,J=5.1, 14.7 Hz), 3.97 (1H, dd, J=5.5, 8.8 Hz), 4.21 (1H, d, J=5.5 Hz),7.19-7.31 (5H, m).

HRMS calcd for C₁₇H₂₁N₂O₆, 349.1400. Found, 349.1388.

Anal. Calcd for C₁₇H₂₁N₂O₆.HCl.H₂O: C, 50.69; H, 5.76; N, 6.95; Cl,8.80. Found: C, 50.66; H, 5.65; N, 6.85; Cl, 8.20.

MS found 349.0 [M+H]⁺.

EXAMPLE 29(1S,2S,4S,5R,6R)-2-(2′S-Amino-4′-carbamoyl-butyrylamino)₄-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Prepare the title compound by dissolving(1S,2S,4S,5R,6R)-2-[2′-tert-butoxycarbonylamino-4′-(trityl-carbamoyl)-butyrylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (1.18 mmoles, Preparation 30) in THF (10 mL) andtreating the solution with 2.5 N LiOH (12 mL). Allow the solution tostir 3 hours and then adjust to pH=2 with 2.5 N HCl. Extract the productfour times with ethyl acetate, dry over anhydrous sodium sulfate andconcentrate in-vacuo to give a white powder. Dissolve the di-acidproduct (0.74 mmol) in 1,2-dichloroethane (3.7 mL) and treat withanisole (0.3 mL) and trifluoroacetic acid (3.72 mL). Stir the reactionmixture at 23° C. for 2.5 hours and then concentrate in-vacuo to give abrown oil. Dissolve the brown oil in water, extract five times withdichloromethane, concentrate the aquesous layer in-vacuo, treat with 1 NHCl (0.74 mL), and lyophilize. Treat the solid with another portion of 1N HCl (2 mL) and lyophilize again to afford 446 mg (quant) of the titlecompound.

[α]_(D) ²³=+8.16 (c=0.49, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.60 (1H, t, J=3.3 Hz), 1.79 (1H, dd, J=5.5,15.4 Hz), 2.10-2.17 (3H, m), 2.46-2.55 (3H, m), 2.60 (1H, dd, J=2.9, 5.9Hz), 3.94 (1H, t, J=6.2 Hz), 4.33 (1H, d, J=5.5 Hz).

MS found 330.0 [M+H]⁺, 351.9 [M+Na]⁺.

HRMS calcd for C₁₃H₁₉N₃O₇, 330.1301. Found, 330.1295.

EXAMPLE 30(1S,2S,4S,5R,6R)-2-(2′S,6′-Diamino-hexanoylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid bis hydrochloride

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S,6′-bis-tert-butoxycarbonylamino-hexanoylamino)₄-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 31).

¹H NMR (400 MHz, CD₃OD) δ 1.47-1.59 (3H, m), 1.66-1.78 (2H, m), 1.84(1H, dd, J=5.4, 15.2 Hz), 1.91 (2H, m), 2.16 (1H, dd, J=3.4, 5.7 Hz),2.45 (1H, d, J=15.7 Hz), 2.66 (1H, dd, J=2.9, 5.9 Hz), 2.97 (2H, t,J=7.3 Hz), 3.92 (1H, t, J=6.4 Hz), 4.33 (1H, d, J=5.4 Hz).

MS found 330.0 [M+H]⁺.

EXAMPLE 31(1S,2S,4S,5R,6R)-2-(2′S-Amino-3′-carbamoyl-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Prepare the title compound by dissolving(1S,2S,4S,5R,6R)-2-[2′-tert-butoxycarbonylamino-3′-(trityl-carbamoyl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (1.07 mmoles, Preparation 32) in THF (12 mL) andtreating the solution with 2.5 N LiOH (12 mL). Allow the solution tostir 3 hours and adjust to pH=2 with 2.5 N HCl. Extract the product fourtimes with ethyl acetate, dry over anhydrous sodium sulfate andconcentrate in-vacuo to give a white powder. Dissolve the di-acidproduct (1.22 mmol) in 1,2-dichloroethane (6 mL) and treat with anisole(1 mL) and trifluoroacetic acid (6 mL). Allow the reaction mixture tostir at 23° C. for 10 hours then concentrate iii-vacuo to give a brownoil. Dilute the brown oil with diethyl ether and the productcrystallized out as a white solid. Filter and wash with copious portionsof ether. Dissolve the solid in a minimal amount of water, treat with0.5 N HCl (5 mL), and lyophilize 3 times to afford 386 mg (90%) of thetitle compound.

[α]_(D) ²³=0 (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.59 (1H, t, 3.4 Hz), 1.76 (1H, dd, J=5.9,16.1 Hz), 2.11 (1H, dd, J=2.9, 5.9 Hz), 2.53 (1H, d, J=16.1 Hz), 2.55(1H, dd, J=2.9, 5.9 Hz), 2.74 (1H, dd, J=9.8, 17.1 Hz), 2.95 (1H, dd,J=3.9, 17.1 Hz), 4.19 (1H, dd, J=3.9, 9.8 Hz), 4.32 (1H, d, J=5.9 Hz).

MS found 316.0 [M+H]⁺, 337.9 [M+Na]⁺.

HRMS calcd for Cl₂H₁₆N₃O₇Na, 338.0964. Found, 338.0953.

EXAMPLE 32(1S,2S,4S,5R,6R)-2-[2′S-Amino-3′-(1H-indol-3-yl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Prepare by dissolving2-[2′-tert-butoxycarbonylamino-3′-(1′-tert-butoxycarbonyl-1H-indol-3′-yl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (0.93 mmoles, Preparation 33) in THF (11 mL) andtreating the solution with 2.5 N LiOH (11 mL). Allow the solution tostir 3 h and then adjust to pH=2 with 2.5 N HCl. Extract the productfour times with ethyl acetate, dry over anhydrous sodium sulfate andconcentrate in-vacuo to give a white powder. Dissolve the di-acidproduct (0.78 mmol) in a solution of 4M HCl in dioxane (20 mL) and allowthe reaction mixture to stir for 3 hours at 23° C. Concentrate thereaction mixture, dilute with dichloromethane, and concentrate againin-vacuo. Dissolve the crude product in methanol, apply to a radialchromatography (silica gel) plate, allowe to spin dry, and elute withMeOH (10%)/NH₄OH (1%)/CHCl₃ to MeOH (60%)/NH₄OH (2%)/CHCl₃. Dissolve theproduct in water and lyophilize two times to afford 139 mg (46%) of thetitle compound.

[α]_(D) ²³=+19.23 (c=0.52, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.43 (1H, t, J=2.9 Hz), 1.57 (1H, dd, J=5.9,15.2 Hz), 2.05 (1H, dd, J=3.4, 6.4 Hz), 2.41 (1H, d, J=14.7 Hz), 2.46(1H, dd, J=2.4, 5.9 Hz), 3.15 (1H, dd, J=5.4, 14.7 Hz), 3.49 (1H, dd,J=5.4, 14.7 Hz), 4.05 (1H, dd, J=5.4, 9.3 Hz), 4.17 (1H, d, J=5.4 Hz),7.09 (2H, m), 7.27 (1H, s), 7.37 (1H, d, J=8.3 Hz), 7.75 (1H, d, J=7.8Hz).

MS found 388.0 [M+H]⁺, 409.9 [M+Na]⁺.

HRMS calcd for C₁₉H₂₁N₃O₆, 388.1508. Found, 388.1502.

EXAMPLE 33(1S,2S,4S,5R,6R)-4-hydroxy-2-[(pyrrolidine-2′S-carbonyl)-amino]-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride salt

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(1′-tert-butoxycarbonyl-pyrrolidine-2′S-carbonyl)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 34), with the exception that thetert-butoxycarbonyl protecting group is removed by treating with 4M HClin dioxane.

[α]_(D) ²³=−4° (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.59 (1H, t, J=3 Hz), 1.78 (1H, dd, J=5.9,15.3 Hz), 2.06 (4H, m), 2.42 (1H, m), 2.59 (2H, m), 3.35 (m, 3H), 4.27(1H, m), 4.32 (1H, d, J=5.9 Hz).

HRMS calcd for C₁₃H₁₉N₂O₆, 299.1243. Found, 299.1242.

Anal. Calcd for C₁₃H₁₈N₂O₆.HCl.H₂O: C, 44.26; H, 6.00; N, 7.94. Found:C, 44.13; H, 5.78; N, 7.62.

EXAMPLE 34(1S,2S,4S,5R,6R)-2-[2′S-Amino-3′-(4-hydroxy-phenyl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride salt

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-[2′S-tert-Butoxycarbonylamino-3′-(4-tert-butoxycarbonyloxy-phenyl)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 35), with the exception that thetert-butoxycarbonyl protecting group was removed by treating with 4M HClin dioxane. Yield 199 mg (50%).

[α]_(D) ²³=+8 (c=0.5, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.48 (1H, t, J=3 Hz), 1.62 (1H, dd, J=5.9,15.3 Hz), 2.03 (1H, m), 2.38 (1H, d, J=15.3 Hz), 2.55 (1H, m), 2.8 (1H,dd, J=8.9, 14.4 Hz), 3.12 (1H, dd, J=5.4, 14.4 Hz), 3.90 (1H, q, J=5.4Hz), 4.21 (1H, d, J=5.9 Hz), 6.7 (2H, d, J=8.4 Hz), 7.05 (2H, d, J=8.4Hz).

HRMS calcd for C₁₇H₂₁N₂O₇, 365.1349. Found, 365.1374.

Anal. Calcd for C₁₇H₂₀N₂O₇.1.1HCl.1.1H₂O: C, 48.12; H, 5.54; N, 6.60.Found: C, 47.89; H, 5.37; N, 6.50.

EXAMPLE 35(1S,2S,4S,5R,6R)-2-(2′S-Amino-4′-methylsufanyl-butyrylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid bis ammonium salt

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S-tert-Butoxycarbonylamino-4′-methylsulfanyl-butyrylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 36), with the exception that thetert-butoxycarbonyl protecting group was removed by treating with 4M HClin dioxane. The final compound was loaded with MeOH onto a 2 mmChromatotron plate and eluted with 50/49.5/0.5 CHCl₃/MeOH/NH₄OH. Theamount of NH₄OH was increased to 1% over the course of the elution togive the zwitterionic compound. Yield 136 mg (37%).

[α]_(D) ²³=+28 (c=1.0, MeOH).

¹H NMR (400 MHz, CD₃OD) δ 1.63 (1H, dd, J=6.0, 15.6 Hz), 2.03 (2H, m),2.12 (3H, s), 2.18 (1H, m), 2.35 (1H, m), 2.49 (1H, d, J=15.6 Hz), 2.65(2H, t, J=8.4 Hz), 3.84 (1H, t, J=6.0 Hz), 4.19 (1H, d, J=6.0 Hz). HRMScalcd for C₁₃H₂₁N₂O₆S, 333.1120. Found, 333.1105.

EXAMPLE 36(1S,2S,4S,5R,6R)-2-[2′S-(2S-Amino-propionylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane)-2,6-dicarboxylicacid

Stir(1S,2S,4S,5R,6R)-2-[2′S-(2S-tert-butoxycarbonylamino-propionylamino)propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic aciddiethyl ester (0.045 g, 0.1 mmol, Preparation 39) in 1 ml of 4N HCl indioxane for 40 min then concentrate the reaction in vacuo. Stir thecrude material in THF (5 ml) and 0.5N LiOH (0.35 mmol) for 1 hour.Adjust the pH=2 with 0.5 N HCl and concentrate the reaction. Purify thecrude product using cation exchange chromatography (Dowex® 50×8-100;elute with 10% pyridine/H₂O) to yield 19 mg (55.3%) of the titlecompound.

¹H NMR (300 MHz, D₂O) δ 1.23 (3H, d, J=7.3 Hz), 1.36 (3H, d, J=7.3 Hz),1.40-145 (1H, m), 1.50 (1H, dd, J=5.5, 15.4 Hz), 1.90-1.96 (1H, m), 2.20(1H, dd, J=2.6, 5.9 Hz), 2.29 (1H, d, J=15.4 Hz), 3.90 (1H, q, J=7.3Hz), 4.15-4.22 (2H, m).

MS (ES) m/z 342.1 [M−1]⁻.

HRMS calcd for C₁₄H₂₂N₃O₇, 344.1458. Found, 344.1457.

EXAMPLE 37(1S,2S,4S,5R,6R)-2-[2′S-(2-Amino-acetylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid

(1S,2S,4S,5R,6R)-2-[2′S-(2-tert-butoxycarbonylamino-acetylamino)-propionylamino]4-hydroxy-bicyclo[3.1.0.]hexane-2,6-dicarboxylicacid diethyl ester (0.12 g, 0.25 mmol, Preparation 40) in THF (5 ml) and1 N LiOH (1.0 mmol) for 1 hour. Adjust the mixture to pH=2 using 1N HCland concentrate. Stir the crude material in a saturated HCl (g) ethylacetate solution at 0° C. Remove excess HCl (g) by purging with N₂ andconcentrate the reaction. Purify the crude product using cation exchangechromatography (Dowex® 50×8-100; elute with 10% pyridine/H₂O) to yield0.06 g (72.9%) of the title compound.

[α]_(D) ²³=−42.11 (c=0.57, H₂O).

¹H NMR (300 MHz, D₂O) δ 1.21 (3H, d, J=7.3 Hz), 1.39 (1H, m), 1.50 (1H,dd, J=5.7, 15.8 Hz), 1.91 (1H, m), 2.15 (1H, dd, J=2.6, 5.9 Hz), 2.28(1H, d, J=15.0 Hz), 3.65 (2H, s), 4.14 (1H, app d, J=5.9 Hz), 4.20 (H,app q, J=7.3 Hz).

HRMS calcd for C₁₃H₂₀N₃O₇, 330.1301. Found, 330.1299.

EXAMPLE 38(1S,2S,4S,5R,6R)-2-[2′S-(2S-Amino-4-methyl-pentanoylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-butoxycarbonylamino-4-methyl-pentanoylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 41). Yield 0.21 g (57%, 0.50 mmoles) ofa white solid.

[α]_(D) ²³=−3.64° (c=0.55, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 0.89 (3H, d, J=4.4 Hz), 0.91 (3H, d, J=4.0Hz), 1.29 (3H, d, J=7.0 Hz), 1.52 (1H, t, J=3.3 Hz), 1.55-1.67 (4H, m),1.97 (1H, dd, J=2.9, 5.9 Hz), 2.38 (1H, dd, J=2.9, 5.9 Hz), 2.45 (1H, d,J=15.4 Hz), 3.75-3.80 (1H, m), 4.18 (1H, d, J=5.9 Hz), 4.33 (1H, app. q,J=7.0 Hz),

Anal Calcd for C₁₇H₂₇N₃O₇.1.0HCl.1.6H₂O: C, 45.30; H, 6.98; N, 9.32; Cl,7.87. Found: C, 44.95; H, 6.54; N, 9.12; Cl, 7.53.

HRMS (ES) calcd for C₁₇H₂₈N₃O₇ [M+H]⁺, 386.1927. Found, 386.1911.

EXAMPLE 39(1S,2S,4S,5R,6R)-2-[2′S-(2-Amino-3-methyl-butyrylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-[2′-(2S-tert-Butoxycarbonylamino-3-methyl-butyrylamino)-propionylamino]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (Preparation 42). Yield 0.15 g (88%, 0.37 mmoles) ofa white solid.

[α]_(D) ²³=−15.69° (c=0.51, MeOH).

¹H NMR (300 MHz, CD₃OD) δ 0.92 (3H, d, J=7.0 Hz), 0.95 (3H, d, J=7.0Hz), 1.28 (3H, d, J=7.0 Hz), 1.52 (1H, t, J=2.9 Hz), 1.60 (1H, dd,J=5.9, 15.4 Hz), 1.98 (1H, dd, J=3.3, 5.9 Hz), 2.03-2.15 (1H, m), 2.39(1H, dd, J=2.9, 6.2 Hz), 2.45 (1H, d, J=15.4 Hz), 3.56 (1H, d, J=5.5Hz), 4.18 (1H, d, J=5.9 Hz), 4.33 (1H, app. q, J=7.0 Hz), 8.82 (1H, s).

Anal Calcd for C₁₆H₂₅N₃O₇.1.0HCl.1.5H₂O: C, 44.19; H, 6.72; N, 9.66; Cl,8.15. Found: C, 44.32; H, 6.48; N, 9.14; Cl, 7.66.

HRMS (ES) calcd for C₁₆H₂₆N₃O₇ [M+H]⁺, 372.1771. Found, 372.1758.

EXAMPLE 40(1R,2S,4R,5R,6R)-2-(2′S-2′-Aminopropionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate monohydrate

Add 16 mL of water followed by 11.4 mL (175 mmol) of methanesulfonicacid to a hazy solution of(1R,2S,4R,5R,6R)-2-(2′S-2′-(tert-butoxycarbonylamino)propionyl)amino-4-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid (32.8 g, 87.5 mmol, Preparation 52) in 295 mL of acetone at gentlereflux dropwise to give a clear, faint yellow solution which yields aslurry after 5 minutes. Stir for 130 minutes, remove the heat source,and add another 295 mL of acetone over 30 minutes. Cool the slurry toroom temperature, then stir an additional 2 hours. Filter the slurry,wash with acetone (2×82 mL), and vacuum dry at 45° C. for 16 hours togive 31.5 g (93%) of the title compound as a white solid.

The material may be further recrystallized. Mix 30.5 g of the abovecrude product with 152.5 mL of acetone and 35 mL of water. Heat to 55°C., and add water (3.66 mL) to effect complete dissolution. Dilute thesolution with 61 mL of acetone and optionally seed. Remove the heatsource and allow the mixture to gradually cool until good nucleation hadcommenced. Add 396 mL of acetone to the slurry over 70 minutes, thenstir at room temperature for an additional 3 hours. Filter, wash withacetone (3×91 mL), and vacuum dry at 45° C. several hours (typicallyovernight) to yield 27.7 g (91% recovery) of the title compound as awhite solid.

mp (DSC) 200° C.

[α]²⁵ _(D) +34° (c 1.0, CH₃OH)

400 MHz ¹H NMR (DMSO-d₆) δ 12.76 (br s, 2H), 9.18 (s 1H), 8.07 (br s,3H), 5.50-5.36 (m, 1H), 3.87 (d, 1H, J=6.8 Hz), 2.82 (dd, 1H, J=14, 8.0Hz), 2.38 (s, 3H), 2.25 (m, 2H), 1.96 (t, 1H, J=3.0 Hz), 1.39 (m, 1H),1.37 (d, 3H, J=6.8 Hz).

100 MHz ¹³C NMR (DMSO-d₆) δ 173.1, 172.3, 169.7, 92.8 (d, C—Fsplitting), 63.2 (d, C—F splitting), 48.0, 39.7, 36.4 (d, C—Fsplitting), 32.5(d, C—F splitting), 29.3 (d, C—F splitting), 19.3 (d,C—F splitting), 16.9.

FTIR (KBr) 3461 (w), 3379 (w), 3269 (m), 2653 (s), 2591 (s), 2529 (s),1724 (s), 1691 (s), 1353 (m), 1287 (s), 1271 (s), 1256 (s), 1212 (s),1147 (s), 1052 (s), 1024 (s), 787 (m) cm⁻¹.

Anal. Cald for C₁₂H₂₁FN₂O₉S: C, 37.11; H, 5.45; N, 7.21. Found: C,37.12; H, 5.45; N, 7.16.

EXAMPLE 41 (1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid monohydrate

Heat a slurry of(1R,4S,5S,6S)-4-(2′S,4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid monosodium salt (110.04 g, 225.3 mmol, Preparation 55) in acetone(110 mL) and water (550 mL) to 55° C. Add concentrated hydrochloric acid(56 mL, 675.8 mmol) dropwise to the stirring slurry to gradually afforddissolution. Upon completion of the addition, stir the solution at 55°C. for 2 hours. Remove the heat source and allow the solution to cool toroom temperature. Filter the solution and rinse with 20 mL of water.Slowly add 2N sodium hydroxide (165 mL, 330 mmol) to the solution toraise pH to 1.71 at which time precipitation occurs. Stir for 10minutes; a thin slurry forms and the pH drops to 0.98. Add more 2Nsodium hydroxide (62 mL, 124 mmol) to the resultant slurry to raise thepH to 3.06 followed by stirring for 3 hours giving a final pH of 3.24.Filter the slurry, wash with water (2×165 mL), and dry at 45° C. for 15hours to afford 73.27 g (85% weight yield) of the title compound as awhite solid.

mp (DSC) 203° C.

[α]²⁵ _(D) +13.4 (c 1.19, 1N HCl).

500 MHz ¹H NMR (D₂O) δ 3.99 (t, 1H, J=6.0 Hz), 3.93 (d, 1H, J=15.0 Hz),3.50 (dd, 1H, J=1.0, 4.0 Hz), 3.12 (d, 1H, J=15.0 Hz), 2.95 (dd, 1H,J=4.0, 7.0 Hz), 2.48 (t, 2H, J=8.0 Hz), 2.33 (t, 1H, J=4.0), 2.09-1.98(m, 5H).

¹³C NMR (125 MHz, D₂O) δ 173.50, 172.60, 169.18, 61.66, 54.76, 52.19,42.55, 31.70, 30.10, 28.09, 23.53, 14.14.

FTIR (ATR) 3558.54 (s), 3024.05 (s), 2959.87 (s), 1748.83 (s), 1692.89(s), 1681.99 (s), 1617.50 (s), 1567.63 (s), 1497.65 (s), 1314.11 (s),1282.22 (s), 1263.26 (s), 1239.01 (s), 1101.46 (s), 884.62 (s), 809.95(s), 773.46 (s) cm⁻¹.

Anal. Cald for C₁₂H₁₈N₂O₇S₂.H₂O: C, 37.49; H, 5.24; N, 7.29. Found: C,37.34; H, 5.04; N, 7.15.

EXAMPLE 42(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid tosylate

Heat a mixture of(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0)hexane-4,6-dicarboxylicacid (118.09 g corrected, 253 mmol, Preparation 56) andp-toluenesulfonic acid monohydrate (54 g, 278 mmol) in toluene (1180 mL)to 75° C., which results in a thick slurry. Stir the slurry at refluxfor 165 minutes. Remove the heat source and allow the slurry to cool toroom temperature followed by stirring overnight. Filter the slurry, washwith toluene (3×240 mL), and dry in vacuo at 45° C. for 22 hours toprovide 134.92 g (98% yield) of the title compound.

mp (DSC) 255° C.

[α]²⁵ _(D) +8.3 (c 1.2, CH₃OH).

500 MHz ¹H NMR (CD₃OD) δ7.71 (d, 2H, J=8.0 Hz), 7.24 (d, 2H, J=8.0 Hz),4.14 (d, 1H, J=15 Hz), 4.00 (t, 1H, J=6.0 Hz), 3.54 (dd, 1H, J=4.0, 7.0Hz), 3.13 (d, 1H, J=15 Hz), 3.01 (dd, 1H, J=4.0, 7.0 Hz), 2.60 (t, 2H,J=8.0 Hz), 2.49 (t, 1H, J=4.0 Hz), 2.37 (s, 3H), 2.19-2.12 (m, 5H).

¹³C NMR (125 MHz, CD₃OD) δ 170.49, 169.69, 168.99, 142.18, 140.67,182.73, 125.79, 60.26, 54.76, 52.21, 42.44, 30.90, 30.77, 27.20, 22.33,20.17, 13.96.

FTIR (ATR) 3091.19 (w), 1730.91 (s), 1668.22 (s), 1563.97 (s), 1518.49(s), 1312.69 (m), 1247.46 (s), 1212.05 (s), 1156.33 (s), 1123.09 (s),1035.95 (s), 1011.36 (s), 892.41 (s), 814.02 (s), 683.69 (s) cm⁻¹.

EXAMPLE 43(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid mesylate

Heat a slurry of(1R,4S,5S,6S)-4-(2′S-4′-methylthio-2′-(tert-butoxycarbonyl)aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid (1.08 g, 2.31 mmol, Preparation 56) in 13 mL of propionitrile to85° C. Add water (540 μL) to the slurry, then add methanesulfonic aciddropwise (225 μL, 3.47 mmol) Stir the slurry for 90 minutes. Remove theheat source and add propionitrile (30 mL). Cool the slurry to roomtemperature and stir for 90 minutes. Filter, wash with propionitrile(3×2.7 mL), and dry at 45° C. overnight to give 1.04 g (97%) of thetitle compound.

mp (DSC) 244° C.

[α]²⁵ _(D) +10.2 (c 1.16, CH₃OH).

500 MHz ¹H NMR (CD₃OD) δ 4.16 (d, 1H, J=15 Hz), 4.00 (t, 1H, J=6.0 Hz),3.54 (dd, 1H, J=4.0, 7.0 Hz), 3.15 (d, 1H, J=15 Hz), 3.01 (dd, 1H,J=4.0, 7.0 Hz), 2.71 (s, 3H), 2.61 (t, 2H, J=8.0), 2.51 (t, 1H, J=4.0),2.20-2.14 (m, 5H).

¹³C NMR (125 MHz, CD₃OD) 3170.50, 169.71, 169.00, 60.27, 54.78, 52.18,42.43, 38.35, 30.90, 30.78, 28.20, 22.35, 13.96.

FTIR (ATR) 3055.57 (m), 1725.90 (s), 1693.60 (s), 1527.33 (s), 1528.96(s), 1320.89 (s), 1176.86 (s), 1152.70 (s), 1118.55 (s), 1051.42 (s),816.49 (s), 786.63 (s) cm⁻¹.

Anal. Cald for C₁₂H₁₈N₂O₇S₂.CH₄O₃S: C, 33.76; H, 4.79; N, 6.06. Found:C, 33.98; H, 4.82; N, 5.98.

EXAMPLE 44(1R,2S,5R,6R)-2-(2′R-aminopropionyl)amino-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Add 1.13 mL (2.8 mmol) of 2.5 N LiOH to a solution of 0.20 g (0.47 mmol)of Isomer A from Preparation 60 in THF (1.2 mL) and stir the resultingmixture overnight at room temperature. Wash the solution with ether,neutralize at 0° C. with 1N HCl, and extract with EtOAc (3×3 mL). Drythe combined organic layers over anhydrous MgSO₄ and concentrate underreduced pressure. Dissolve the crude in 3.76 mL of 1N HCl in EtOAc andstir overnight. Decant the solvent, wash with ether, and dry the productwith an Ar stream to give the title compound (83 mg, 57% yield) as awhite solid.

¹H-NMR (300 MHz, CDCl₃): 1.28 (d, 3H. J=7.1 Hz), 1.64-1.69 (m, 1H),1.86-2.02 (m, 1H), 2.11-2.21 (m, 3H), 2.54 (d, 1H, J=7.1 Hz), 3.84 (q,1H, J=7.1 Hz).

EXAMPLE 45(1S,2R,5S,6S)-2-(2′R-aminopropionyl)amino-6-fluoro-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Beginning with a solution Isomer B of Preparation 60, the title compoundis prepared essentially as Example 44.

¹H-NMR (300 MHz, CDCl₃): 1.30 (d, 3H, J=7.1 Hz), 1.59-1.65 (m, 1H),1.85-2.03 (m, 1H), 2.05-2.25 (m, 3H), 2.53 (d, 1H, J=6.6 Hz), 3.85 (q,1H, J=7.1 Hz).

EXAMPLE 46(1S,2S,4S,5R,6R)-2-(2′S-aminopropionyl)amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride

Prepare according to General Procedure C using(1S,2S,4S,5R,6R)-2-(2′S-tert-butoxycarbonylamino-propionylamino)-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid diethyl ester (0.47 g, 1.1 mmol, Preparation 37) to yield 0.23 g(67.7%) of the title compound.

[α]_(D) ²³=−4.1 (c=0.49, MeOH).

¹H NMR (CD₃OD) δ 1.51 (3H, d, J=7.0 Hz), 1.56 (1H, t, J=2.9 Hz), 1.77(1H, dd, J=5.8, 15.8 Hz), 2.1-2.13 (1H, m), 2.52 (1H, d, J=16.1 Hz),2.57 (1H, dd, J=2.9, 5.9 Hz), 3.88 (1H, dd, J=7.0, 14.3 Hz), 4.32 (1H,d, J=5.5 Hz).

Anal. Calcd for C₁₁H₁₇N₂O₆.1.1HCl.0.9H₂O: C, 40.12; H, 5.80; N, 8.53.Found: C, 39.85; H, 5.41; N, 8.36.

HRMS calcd for C₁₁H₁₆N₂O₆Na, 295.0906. Found, 295.0883.

Prodrug compounds of the present invention may be evaluated against thecorresponding parent compound through various cellular uptake assays.These assays can provide comparative data to permit one of ordinaryskill in the art to identify compounds which are readily absorbed intothe cell to provide superior exposure. Two such assays include theGly-Sar Uptake Assay and the Caco-2 Assay, described below.

Gly-Sar Uptake Assay

It has been realized that some orally administered peptidomimetic drugsare absorbed through the intestinal peptide transport system. Yang, etal., Pharm. Res. 16(9) (1999). In particular, the intestinal peptidetransporter hPepT1 has been studied for its expression of inhibition ofpeptidyl uptake and its corresponding level of recognition within acell. Meredith, et al, Eur. J. Biochem., 267, 3723-3728 (2000). Further,characterizing the intestinal absorption mechanism of amino acids in thehPepT1 transporter has been targeted as an effective strategy foridentifying improved oral drug absorptions. Han, et al., Polym. Prepr.(Am. Chem. Soc., Div. Polym. Chem) 40(1): 259-260 (1999); Sawada, etal., J. Pharmacol. Exp. Ther., 291(2): 705-709 (1999).

U.S. Pat. No. 5,849,525 describes methods which could be used to measurethe level of affinity of compounds of the present invention with thehPepT1 transporter.

For example, stably transfected Chinese Hamster Ovary (CHO) cellsover-expressing the hPepT1 transporter could be used to test compoundsof the present invention. The CHO cells would monitored for the uptakeof Gly-Sar, which when uptake in the presence of the prodrug compoundsof the present invention is in amounts greater than when the cell isfree from prodrug compounds of the present invention would be indicativeof agonist activity; and which when uptake of the prodrug compounds ofthe present invention is less than the uptake in the absence of prodrugcompounds of the present invention would be indicative of inhibitoryactivity. Compounds of the present invention generally exhibit EC₅₀values of less than 5 mM.

Caco-2 Assay

Another particular method for measuring the uptake of compounds of thepresent invention into cells is to study the peptide transport carrierof human intestinal cell line Caco-2. Human adenocarcinoma cells(Memorial Sloan-Kettering Cancer Center, Rye, N.Y., and/or ATCC,Rockville, Md.) are passaged in Dulbecco's Modified Eagle mediumcontaining 10% fetal calf serum and 1% Minimal Essential Medianon-essential amino acid solution without addition of sodium pyruvate orantibiotics. These cells were mycoplasma-free and were used betweenpassage numbers 28 and 40. For flux measurements, between 5 to 10×10⁴cells are grown in collagen-coated multiwell dishes for 13-18 days andthe medium is replaced every two to three days.

Drug uptake is measured at 37° C. using a test compound employing acluster-tray technique (see Gazzola, et al., Anal. Biochem. 115, 368-74(1981)). The flux buffer is bicarbonate-free Earle's balanced saltsolution containing 25 mM Mes titrated to pH 6.0 with KOH, and cholinechloride in place of sodium chloride. The osmolality of the flux bufferis adjusted to 300±5 mosmol/kg with choline chloride. [³H]Inulin is usedas a marker for the extracellular fluid that adheres to cells during thewashing procedure to estimate the zero time for determining the rate ofuptake. Fresh solutions of the test compounds and dipeptides areprepared daily. At the end of the experiment cells, are lysed in water,test compounds can be detected in cell lysates using LC/MS/MS. Proteinis measured by the method described in Smith, et al., Anal. Biochem.150, 76-85 (1985).

Uptake is measured over a 40 minute. Initial uptake rates are calculatedin the linear region of the time course regression and an estimated zerotime as described above using linear regression. Percent inhibition iscalculated based on the control uptake rate measured in the absence of adipeptide. For examples of this Caco-2 assay, see Dantzig & Bergin,Biochim. Biophys. Acta 1027, 211-17 (1990).

In Vivo Exposure as Measured by Rat Plasma Concentration

To study the in vivo exposure of compounds of Formula II following oraldosing of compounds of Formula I in comparison to compounds of FormulaII, studies measuring the plasma concentrations of the respectivecompound of Formula II in rats are performed. Mature Fischer 344 malerats (190-270 gram) are obtained from Harlan Sprague-Dawley, Cumberland,Ind., USA, and acclimated in the study housing for 3 days. On day 4,test compounds are dissolved in buffered water (1 mg/ml=test compound/20mM potassium dihydrogen phosphate, pH=2) and given orally as a single 5mg/kg dose. Blood samples are collected through orbital sinus or cardiacpuncture (last time point) at 0.5 and 1 hour or, alternatively, at 1 and3 hours. Plasma samples are stored at −20° C. in the presence ofphenylmethylsulfonyl fluoride, a protease inhibitor, prior to analysis.Plasma samples and internal standard compounds are pretreated by solidphase extraction (SAX support, methanol/water/dilute acetic acid).

As shown in Tables 1A and 1B below, the plasma concentrations (ng/ml) ofthe respective compound of Formula II for each test compound aredetermined by LC/MS/MS and are presented as a sum of the concentrationsat the 0.5 and 1 hour or, alternatively, at the 1 and 3 hour sample timepoints. TABLE 1A In Vivo Exposure Assay Compound Rat Exposure (ng/ml of(1R,4S,5S,6S)-4-(2′S- Aminopropionyl)amino-]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0.]hexane- 4,6-dicarboxylic acid hydrochloride)Example 1 2251 ng/ml (following 10 mg/kg p.o.) Non-prodrug form of 1521ng/ml (following 5 mg/kg p.o.) Example 1 3981 ng/ml (following 10 mg/kgp.o.)

TABLE 1B In Vivo Exposure Assay Compound Rat Exposure (ng/ml of(1R,2S,4R,5R,6R)-2-(2′S- Amino-propionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6- dicarboxylic acid hydrochloride) Example14 5271 ng/ml (following 5 mg/kg p.o.) Non-prodrug form of 1162 ng/ml(following 5 mg/kg p.o.) Example 14 1342 ng/ml (following 10 mg/kg p.o.)

As shown above in Tables 1A and 1B, when given orally to rats, thecompounds of the current invention exhibit comparable plasmaconcentration of the parent compound. This demonstrates compounds of thepresent invention are converted to the parent compounds, compounds ofFormula II, in vivo.

The compounds of the present invention are preferably formulated priorto administration. Therefore, another aspect of the present invention isa pharmaceutical formulation comprising a compound of Formula I, or apharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier, diluent, or excipient. Thepharmaceutical formulations may be prepared by procedures well-known byone of ordinary skill in the art. In making the compositions of thepresent invention, the active ingredient will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier, and maybe in the form of a capsule, sachet, paper, or other container. When thecarrier serves as a diluent, it may be a solid, semi-solid, or liquidmaterial which acts as a vehicle, excipient, or medium for the activeingredient. The compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols, ointments containing, for example, up to10% by weight of active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum, acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc,magnesium stearate, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents, or flavoring agents.Compositions of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well-known in theart.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 mg to about 500 mg active ingredient,preferably about 25 mg to about 300 mg active ingredient. As used hereinthe term “active ingredient” refers to a compound included within thescope of Formula I.

The term “unit dosage form” refers to a physically discrete unitsuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical carrier, diluent, or excipient.

1-37. (canceled)
 38. A compound of Formula I

wherein: A is H-(Q)_(p)-; Q is independently selected, each time taken,from the group amino acyl; p is an integer from 1 to 10; X is O, S, SO,SO₂, or CR³R⁴; R³ is fluoro, X′OR⁵, SO₃H, tetrazol-5-yl, CN, PO₃R⁶ ₂,hydroxy, NO₂, N₃, (CH₂)_(m)COOR^(5a), (CH₂)_(m)PO₃R^(6a) ₂,NHCONHR^(5b), or NHSO₂R^(5c) and R⁴ is hydrogen; or R³ and R⁴ eachrepresent fluoro; or R³ and R⁴ together represent ═O, ═NOR⁷, ═CR⁸R⁹,═CHCOOR^(5b), ═CHPO₃R^(6a) ₂, or ═CHCN; or one of R³ or R⁴ representsamino and the other represents carboxyl; X′ represents a bond, CH₂, orCO; m is an integer from 1 to 3; R⁵, R^(5a), R^(5b), R^(5c), R⁷, R⁸, andR⁹ are independently a hydrogen atom; an optionally substituted (1-6C)alkyl group; an optionally substituted (2-6C) alkenyl group; anoptionally substituted (2-6C) alkynyl group; an optionally substitutedaromatic group; an optionally substituted heteroaromatic group; anon-aromatic carbocyclic group; a non-aromatic heterocyclic group; anon-aromatic monocyclic carbocyclic group fused with one or twomonocyclic aromatic or heteroaromatic groups; or a non-aromaticmonocyclic heterocyclic group fused with one or two monocyclic aromaticor heteroaromatic groups; R⁶ and R^(6a) independently represent hydrogenor a (1-6C)alkyl group; R¹⁰ is hydrogen or fluoro; and R¹¹ is hydrogen,fluoro, or hydroxy; or a pharmaceutically acceptable salt thereof.
 39. Acompound or salt according to claim 38, provided that the compound orsalt is not one in which X is CR³R⁴ wherein R³ is fluoro and R⁴ ishydrogen, p is 1, and Q is L-alanyl; or a pharmaceutically acceptablesalt thereof.
 40. A compound or salt according to claim 38 wherein A isH-(Q)_(p)-; Q is independently selected, each time taken from the groupamino acyl; p is an integer from 1 to 3; X is O, S, SO, SO₂, or CR³R⁴;R³ is fluoro or hydroxy, and R⁴ is hydrogen; or R³ and R⁴ togetherrepresent ═O; R¹⁰ is hydrogen or fluoro; and R¹¹ is hydrogen, fluoro, orhydroxy.
 41. A compound or salt according to claim 39 A is H-(Q)_(p)-; Qis independently selected, each time taken, form the group amino acyl; pis an integer from 1 to 3; X is O, S, SO, SO₂, or CR³R⁴; R³ is fluoro orhydroxy, and R⁴ is hydrogen; or R³ and R⁴ together represent ═O; R¹⁰ ishydrogen or fluoro; and R¹¹ is hydrogen, fluoro, or hydroxy.
 42. Acompound or salt according to claim 38 wherein Q is an amino acylderived from a natural amino acid.
 43. A compound or salt according toclaim 39 wherein Q is an amino acyl derived from a natural amino acid.44. A compound or salt according to claim 40 wherein Q is an amino acylderived from a natural amino acid.
 45. A compound or salt according toclaim 41 wherein Q is an acyl derived from a natural amino acid.
 46. Acompound or salt according to any one of claim 38-45 wherein X is SO₂.47. A compound or salt according to any one of claims 38-45 wherein X isCR³R⁴, R³ is fluoro, and R⁴ is hydrogen.
 48. A compound or saltaccording to any one of claims 38-45 wherein X is CR³R⁴, R³ is hydroxy,and R⁴ is hydrogen.
 49. A pharmaceutically acceptable salt according toclaim 38 that is an acid-addition salt made with an acid which providesa pharmaceutically acceptable anion a base-addition salt made with abase which provides a pharmaceutically acceptable anion for a compoundwhich contains an acidic moiety, or a zwitteronic compound whichcontains oppositely charged groups.
 50. A compound according claim 38wherein A is H-(Q)_(p)-; Q is L-alanyl; p is 1; X is SO₂ or CR³R⁴; R³ isfluoro and R⁴ is hydrogen; R¹⁰ is hydrogen; and R¹¹ is hydrogen; or thehydrochloride salt, tosylate salt, mesylate salt, esylate salt, besylatesalt, or monosodium salt thereof.
 51. The pharmaceutically acceptablesalt according to claim 50 which is(1R,4S,5S,6S)-4-(2′S-Aminopropionyl)amino]-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid hydrochloride or(1R,4S,6S)-4-(2′S-2′-Aminopropionyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid tosylate.
 52. The compound according to claim 38 which is(1R,4S,5S,6S)-4-(2′S,4′-methylthio-2′-aminobutanonyl)amino-2,2-dioxo-2λ⁶-thia-bicyclo[3.1.0]hexane-4,6-dicarboxylicacid or a pharmaceutically acceptable salt thereof.
 53. The compoundaccording to claim 52 which is (1R,4S,5S,6S)-4-(2′S-4′-acid monohydrate.54. The pharmaceutically acceptable salt according to claim 38 that is1S,2S,4S,5R,6R-2-(2′S-aminopropionyl)amino-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride.
 55. A compound according to claim 38 wherein A isH-(Q)_(p)-; Q is L-alanyl; p is 1; X is CR³R⁴; R³ is fluoro and R⁴ ishydrogen; R¹⁰ is hydrogen; and R¹¹ is hydrogen; or a pharmaceuticallyacceptable salt thereof.
 56. The compound or salt according to claim 55which is selected from the group consisting of: a)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid hydrochloride; b)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-carboxylicacid mesylate; c)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid esylate; d)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid besylate; e)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid tosylate; f)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid; and g)1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicmonosodium salt.
 57. The pharmaceutically acceptable salt according toclaim 56 which is1R,2S,4R,5R,6R-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate.
 58. The pharmaceutically acceptable salt according toclaim 57 which is(1R,2S,4R,5R,6R)-2-(2′S-2′-Aminopropionyl)amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylicacid mesylate monohydrate.
 59. A process for preparing a compound ofFormula I, or a pharmaceutically acceptable salt thereof, as claimed inclaim 38 comprising acylating a compound of formula (ii)

with a corresponding amino acyl of Formula IIIPgN-A-  (III) wherein Pg^(N) is a nitrogen-protecting group; whereafterfor any of the above procedures, when a functional group is protectedusing a protecting group, removing the protecting group; whereafter, forany of the above procedures; when a pharmaceutically acceptable salt ofa compound of Formula I is required, reacting the basic form of such acompound of Formula I with an acid affording a pharmaceuticallyacceptable counterion; or for a compound of Formula I which bears anacidic moiety, reacting the acidic form of such a compound of Formula Iwith a base which affords a pharmaceutically acceptable cation; or for azwitterionic compound of Formula I, neutralizing the acid-addition saltfrom or base-addition salt from of such a compound of Formula I; or byany other conventional procedure.
 60. A method for affecting thecAMP-linked metabotrophic glutamate receptors in a patient, whichcomprises administrating to a patient requiring modulated excitatoryamino acid neurotransmission a pharmaceutically effective amount of acompound of claim
 38. 61. A method for affecting the cAMP-linkedmetabotropic glutamate receptors in a patient, which comprisesadministering to a patient requiring modulated excitatory amino acidneurotransmission a pharmaceutically effective amount of a compound ofclaim
 39. 62. A method of administering an effective amount of acompound of Formula II,

wherein X and R¹⁰ are defined as in claim 38, which comprisesadministering to a patient requiring modulated excitatory amino acidneurotransmission a pharmaceutically effective amount of a compound ofclaim
 38. 63. A method of administering an effective amount of acompound of Formula II,

wherein X nd R¹⁰ are defined as in claim 39, which comprisesadministering to a patient requiring modulated excitatory amino acidneurotransmission a pharmaceutically effective amount of a compound ofclaim
 39. 64. A method for treating a neurological disorder in a patientwhich comprises administering to the patient in need of treatmentthereof a pharmaceutically-effective amount of a compound of claim 38.65. A method for treating a neurological disorder in a patient whichcomprises administering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of claim
 39. 66. Themethod of claim 64 wherein said neurological disorder is cerebraldeficits subsequent to cardiac bypass and grafting; cerebral ischemia;spinal cord head trauma; Alzheimer's Disease; Huntington's Chorea;amyotrophic lateral sclerosis; AIDS-induced dementia perinatal hypoxia;hypoglycemic neuronal damage; ocular damage and retinopathy; cognitivedisorders; idiopathic and drug-induced Parkinson's Disease; muscularspasms; migraine headaches; urinary incontinence; drug tolerance,withdrawal, cessation, and craving; smoking cessation; emesis; brainedema; chronic pain; sleep disorders; convulsions; Tourette's syndrome;attention deficit disorder; and tardive dyskinesia.
 67. The method ofclaim 65 wherein said neurological disorder is cerebral deficitssubsequent to cardiac bypass and grafting; cerebral ischemia; spinalcord trauma; head trauma; Alzheimer's Disease; Huntington's Choreaamyotrophic lateral sclerosis; AIDS-induced dementia; perinatal hypoxia;hypoglycemic neuronal damage; ocular damage and retinopathy, cognitivedisorders; idiopathic and drug-induced Parkinson's Disease; muscularspasms; migraine headaches; urinary incontinence; drug tolerance,withdrawal, cession, and craving; smoking cessation emesis; brain edema;chronic pain; sleep disorders; convulsions; Tourette's syndrome;attention deficit disorder; and tardive dyskinesia.
 68. The method ofclaim 66 or 67 wherein said neurological disorder is drug tolerance,withdrawal, cessation, and craving; or smoking cessation.
 69. A methodfor treating a psychiatric disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of claim
 38. 70. Amethod for treating a psychiatric disorder in a patient which comprisesadministering to the patient in need of treatment thereof apharmaceutically-effective amount of a compound of claim
 39. 71. Themethod of claim 69 wherein said psychiatric disorder is schizophrenia,anxiety and related disorder, depression, bipolar disorders, psychosis,and obsessive compulsive disorders.
 72. The method of claim 70 whereinsaid psychiatric disorder is schizophrenia, anxiety and relateddiscords, depression, bipolar disorders, psychosis, and obsessivecompulsive disorders.
 73. The method according to any one of claims 71or 72 wherein said psychiatric disorder is anxiety and relateddisorders.
 74. A pharmaceutical formulation comprising in associationwith a pharmaceutically acceptable carrier, dilutent or excipient, acompound of Formula I, or a pharmaceutically acceptable salt thereof.